ES2344248T3 - SEQUENCES OF NUCLEOTIDES CODING THE DAPC GENE, AND PROCEDURE FOR THE PREPARATION OF L-LISINE. - Google Patents
SEQUENCES OF NUCLEOTIDES CODING THE DAPC GENE, AND PROCEDURE FOR THE PREPARATION OF L-LISINE. Download PDFInfo
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- ES2344248T3 ES2344248T3 ES01103850T ES01103850T ES2344248T3 ES 2344248 T3 ES2344248 T3 ES 2344248T3 ES 01103850 T ES01103850 T ES 01103850T ES 01103850 T ES01103850 T ES 01103850T ES 2344248 T3 ES2344248 T3 ES 2344248T3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- Biochemistry (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Saccharide Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Un polinucleótido aislado, que codifica un polipéptido con actividad de N-succinil-aminocetopimelato transaminasa, que comprende una secuencia de aminoácidos, que es idéntica en por lo menos un 90% a la secuencia de SEQ ID NO.An isolated polynucleotide, which encodes a polypeptide with N-succinyl aminoacetopimelate transaminase activity, comprising an amino acid sequence, which is at least 90% identical to the sequence of SEQ ID NO.
Description
Secuencias de nucleótidos que codifican el gen dapC, y procedimiento para la preparación de L-lisina.Nucleotide sequences that encode the gene dapC, and procedure for the preparation of L-lysine
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Son objeto del invento unas secuencias de nucleótidos que codifican el gen dapC y procedimientos para la preparación por fermentación de L-lisina mediando utilización de bacterias corineformes, en las que es sobreexpresado el gen dapC (gen de N-succinil-aminocetopimelato transaminasa).Sequences of nucleotides that encode the dapC gene and procedures for L-lysine fermentation preparation mediating use of coryneform bacteria, in which it is overexpressed the dapC gene (gene from N-succinyl aminoacetopimelate transaminase).
Ciertos aminoácidos, en particular la L-lisina, encuentran utilización en la medicina humana y en la industria farmacéutica, pero especialmente en la nutrición de animales.Certain amino acids, in particular the L-lysine, find use in medicine human and in the pharmaceutical industry, but especially in the Animal nutrition
Es conocido que se preparan aminoácidos mediante fermentación de cepas de bacterias corineformes, en particular Corynebacterium glutamicum. A causa de la gran importancia, se trabaja constantemente en el mejoramiento de los procedimientos de preparación. Unas mejoras en los procedimientos pueden referirse a medidas técnicas de fermentación tales como p.ej. agitación y abastecimiento con oxígeno, o a la composición de los medios nutritivos tal como p.ej. a la concentración de azúcares durante la fermentación, o al tratamiento para dar la forma del producto mediante p.ej. una cromatografía de intercambio de iones, o las propiedades intrínsecas de rendimiento del propio microorganismo.It is known that amino acids are prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum . Because of the great importance, work is constantly being done to improve the preparation procedures. Improvements in the procedures may refer to technical fermentation measures such as, for example, stirring and supplying with oxygen, or to the composition of the nutrient media such as, for example, the concentration of sugars during fermentation, or to the treatment for give the product shape by means of an ion exchange chromatography, or the intrinsic performance properties of the microorganism itself.
Con el fin de mejorar las propiedades de rendimiento de estos microorganismos se utilizan métodos de la mutagénesis, la selección y la elección de mutantes. De esta manera se obtienen unas cepas, que son resistentes contra anti-metabolitos, tales como p.ej. el compuesto análogo a lisina S-(2-amino-etil)cisteína, o son auxótrofas para metabolitos importantes en cuanto a regulación y producen unos L-aminoácidos tales como p.ej. L-lisina.In order to improve the properties of performance of these microorganisms methods of the mutagenesis, selection and choice of mutants. In this way strains are obtained, which are resistant against anti-metabolites, such as eg the compound lysine analog S- (2-amino-ethyl) cysteine, or they are auxotrophs for important metabolites in terms of regulation and produce L-amino acids such as e.g. L-lysine
Desde hace algunos años, se emplean asimismo métodos de la tecnología del ADN recombinante para el mejoramiento de las cepas de Corynebacterium que producen aminoácidos, amplificando unos genes individuales de la biosíntesis de aminoácidos e investigando la repercusión sobre la producción de los aminoácidos. Unos artículos recopilatorios acerca de esto se encuentran, entre otras, en las citas de Kinoshita ("Glutamic Acid Bacteria" [Bacterias de ácido glutámico], en: Biology of Industrial Microorganisms, de Demain y Solomon (coordinadores de edición), Benjamin Cummings, Londres, Reino Unido, 1985, 115-142), de Hilliger (BioTec 2, 40-44 (1991)), de Eggeling (Amino Acids 6: 261-272 (1994)), de Jetten y Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) y de Sahm y colaboradores (Annuals of the New York Academy of Science 782, 25-39 (1996)).For some years, recombinant DNA technology methods have also been used for the improvement of Corynebacterium strains that produce amino acids, amplifying individual genes of amino acid biosynthesis and investigating the impact on amino acid production. Some compilation articles about this are found, among others, in the quotations of Kinoshita ("Glutamic Acid Bacteria" [Bacteria of glutamic acid], in: Biology of Industrial Microorganisms, Demain and Solomon (editing coordinators), Benjamin Cummings, London, United Kingdom, 1985, 115-142), of Hilliger (BioTec 2, 40-44 (1991)), of Eggeling (Amino Acids 6: 261-272 (1994)), of Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and de Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
Los autores del invento se han establecido como misión la de poner a disposición unas nuevas medidas para la preparación por fermentación mejorada de L-lisina.The inventors have established themselves as mission of making available new measures for the improved fermentation preparation of L-lysine
La L-lisina encuentra utilización en la medicina humana, en la industria farmacéutica y en particular en la nutrición de animales. Existe por lo tanto un interés general en poner a disposición nuevos procedimientos mejorados para la preparación de L-lisina.L-lysine finds use in human medicine, in the pharmaceutical industry and in Particular in animal nutrition. There is therefore a general interest in making new procedures available improved for the preparation of L-lysine.
Cuando en lo sucesivo se mencionan la L-lisina o la lisina, se entienden por estos nombres no solamente la base, sino también las sales tales como p.ej. el monohidrocloruro de lisina o el sulfato de lisina.When hereafter mentioned the L-lysine or lysine, are understood by these names not only the base, but also salts such as eg lysine monohydrochloride or lysine sulfate.
Es objeto del invento un polinucleótido aislado a partir de bacterias corineformes, que contiene por lo menos una secuencia de polinucleótido, seleccionado entre el conjunto formado porThe object of the invention is an isolated polynucleotide from coryneform bacteria, which contains at least one polynucleotide sequence, selected from the set formed by
- a) to)
- un polinucleótido que codifica un polipéptido con actividad de N-succinil-aminocetopimelato transaminasa, que contiene una secuencia de aminoácidos, que es idéntica en por lo menos un 90% a la secuencia de aminoácidos de SEQ ID No. 2, oa polynucleotide encoding a polypeptide with activity of N-succinyl aminoacetopimelate transaminase, which contains an amino acid sequence, which is identical in at least 90% to the amino acid sequence of SEQ ID No. 2, or
- b) b)
- un polinucleótido que es complementario con respecto a los polinucleótidos de a).a polynucleotide that is complementary with respect to to the polynucleotides of a).
Es objeto del invento asimismo el polinucleótido de acuerdo con la reivindicación 1, tratándose de manera preferente de un ADN replicable, que contiene:The object of the invention is also the polynucleotide according to claim 1, being preferably treated of a replicable DNA, which contains:
- (i) (i)
- la secuencia de nucleótidos, que se muestra en SEQ ID No. 1, othe nucleotide sequence, shown in SEQ ID No. 1, or
- (ii) (ii)
- por lo menos una secuencia, que corresponde a la secuencia (i) dentro de la zona de la degeneración del código genético, oat least one sequence, which corresponds to the sequence (i) within the area of code degeneration genetic, or
- (iii) (iii)
- por lo menos una secuencia, que se hibrida con la secuencia complementaria con la secuencia (i) o (ii), y eventualmenteat least one sequence, which hybridizes with the sequence complementary to sequence (i) or (ii), and eventually
- (iv) (iv)
- unas mutaciones de sentido funcionalmente neutras en (i).functionally neutral sense mutations in (i).
Otros objetos del invento sonOther objects of the invention are
- \quadquad
- un polinucleótido de acuerdo con la reivindicación 5, que contiene la secuencia de nucleótidos que se representa en SEQ ID No. 1,a polynucleotide according to claim 5, which contains the nucleotide sequence that is represented in SEQ ID No. 1,
- \quadquad
- un polinucleótido, que codifica un polipéptido, que contiene la secuencia de aminoácidos, como se representa en SEQ ID No. 2,a polynucleotide, which encodes a polypeptide, which contains the amino acid sequence, as depicted in SEQ ID No. 2,
- \quadquad
- un vector, que contiene el polinucleótido de acuerdo con la reivindicación 1, en particular el vector de lanzadera o el vector plasmídico pXT-dapCexp, que se representa en la Figura 2 y se ha depositado bajo en nombre de DSM 13254 en DSM 5715,a vector, which contains the polynucleotide according with claim 1, in particular the shuttle vector or the plasmid vector pXT-dapCexp, which is represented in Figure 2 and has been deposited under the name of DSM 13254 in DSM 5715,
- \quadquad
- y bacterias corineformes que sirven como células anfitrionas, que contienen el vector.and coryneform bacteria that serve as cells hosts, which contain the vector.
Son objeto del invento asimismo unos polinucleótidos, que se componen en lo esencial de una secuencia de polinucleótido, los cuales son obtenibles por escrutinio mediante hibridación de un correspondiente banco de genes, que contienen el gen completo con la secuencia de polinucleótido que corresponde a SEQ ID No. 1, con una sonda, que contiene la secuencia de polinucleótido que se menciona según SEQ ID No. 1 o un fragmento de la misma, y por aislamiento de la mencionada secuencia de ADN.Some objects of the invention are also polynucleotides, which are composed essentially of a sequence of polynucleotide, which are obtainable by scrutiny by hybridization of a corresponding gene bank, which contain the complete gene with the polynucleotide sequence corresponding to SEQ ID No. 1, with a probe, which contains the sequence of polynucleotide mentioned according to SEQ ID No. 1 or a fragment of the same, and by isolation of said DNA sequence.
Las secuencias de polinucleótidos de acuerdo con el invento son apropiadas como sondas de hibridación para ARN, ADNc y ADN, con el fin de aislar un ADNc (ácido desoxirribonucleico complementario) de plena longitud, que codifican la N-succinil-aminocetopimelato transaminasa y aislar tales ADNc o unos genes, que presentan una alta similaridad de su secuencia con la del gen N-succinil-aminocetopimelato transaminasa.The polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate a cDNA (deoxyribonucleic acid complementary) full length, which encode the N-succinyl aminoacetopimelate transaminase and isolate such cDNA or genes, which have a high similarity of its sequence with that of the gene N-succinyl aminoacetopimelate transaminase
Las secuencias de polinucleótidos de acuerdo con el invento son apropiadas además como cebadores para la producción de los ADN de genes que codifican la N-succinil-aminocetopimelato transaminasa, mediante la reacción en cadena de la polimerasa (PCR, acrónimo de Polymerase Chain Reaction).The polynucleotide sequences according to the invention are also suitable as primers for production of the gene DNAs that encode the N-succinyl aminoacetopimelate transaminase, by polymerase chain reaction (PCR, acronym for Polymerase Chain Reaction).
Tales oligonucleótidos, que sirven como sondas o cebadores, contienen por lo menos 30, de manera preferida por lo menos 20, de manera muy especialmente preferida por lo menos 15 nucleótidos consecutivos. Son apropiados asimismo unos oligonucleótidos que tienen una longitud de por lo menos 40 ó 50 nucleótidos.Such oligonucleotides, which serve as probes or primers, contain at least 30, preferably so minus 20, very especially preferably at least 15 consecutive nucleotides. They are also appropriate oligonucleotides having a length of at least 40 or 50 nucleotides
El concepto de "aislado" significa separado desde su entorno natural.The concept of "isolated" means separate from its natural environment.
El concepto de "polinucleótido" se refiere en general a poli(ribonucleótidos) y poli-(desoxirribonucleótidos), pudiéndose tratar de un ARN o ADN no modificado o de un ARN o ADN modificado.The concept of "polynucleotide" refers to in general to poly (ribonucleotides) and poly- (deoxyribonucleotides), being able to treat a non-RNA or DNA modified or from a modified RNA or DNA.
Por el concepto de "polipéptidos" se entienden unos péptidos o unas proteínas que contienen dos o más aminoácidos unidos a través de enlaces peptídicos.By the concept of "polypeptides" is understand peptides or proteins that contain two or more amino acids linked through peptide bonds.
Los polipéptidos de acuerdo con el invento incluyen un polipéptido de acuerdo con SEQ ID No. 2, en particular los que tienen la actividad biológica de la N-succinil-aminocetopimelato transaminasa y también los que presentan una identidad desde por lo menos un 90% hasta un 95% con el polipéptido de acuerdo con SEQ ID No. 2, y la actividad mencionada.The polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those who have the biological activity of the N-succinyl aminoacetopimelate transaminase and also those with an identity as far as minus 90% up to 95% with the polypeptide according to SEQ ID No. 2, and the activity mentioned.
El invento se refiere además a un procedimiento para la preparación por fermentación de L-lisina, mediando utilización de bacterias corineformes, que en particular ya producían un aminoácido, y en las cuales se refuerzan, en particular se sobreexpresan las secuencias de nucleótidos que codifican el gen dapC.The invention further relates to a process. for the preparation by fermentation of L-lysine, mediating use of coryneform bacteria, which in particular they already produced an amino acid, and in which they are reinforced, in particular nucleotide sequences that are overexpressed are overexpressed encode the dapC gene.
El concepto de "refuerzo" describe en este contexto la elevación de la actividad intracelular de una o varias enzimas en un microorganismo, que son codificadas por el correspondiente ADN, aumentando por ejemplo el número de copias del gen o respectivamente de los genes, utilizando un promotor fuerte o utilizando un gen o alelo, que codifica una correspondiente enzima con una alta actividad, y eventualmente se combinan estas medidas.The concept of "reinforcement" described in this context the elevation of the intracellular activity of one or several enzymes in a microorganism, which are encoded by the corresponding DNA, for example increasing the number of copies of the gene or genes respectively, using a strong promoter or using a gene or allele, which encodes a corresponding enzyme with high activity, and eventually these are combined measurements.
Los microorganismos, que son objeto del presente invento, pueden producir L-aminoácidos, en particular L-lisina, a partir de glucosa, sacarosa, lactosa, fructosa, maltosa, melazas, un almidón, una celulosa, o a partir de glicerol y etanol. Puede tratarse de representantes de bacterias corineformes, en particular del género Corynebacterium. En el caso del género Corynebacterium hay que mencionar en particular la especie Corynebacterium glutamicum, que es conocida en el mundo especializado por su capacidad de producir L-aminoácidos.The microorganisms, which are the subject of the present invention, can produce L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, a starch, a cellulose, or from glycerol and ethanol. They may be representatives of coryneform bacteria, in particular of the genus Corynebacterium. In the case of the genus Corynebacterium, the species Corynebacterium glutamicum , which is known in the world specialized in its ability to produce L-amino acids, should be mentioned in particular.
Cepas apropiadas del género Corynebacterium, en particular de la especie Corynebacterium glutamicum, son por ejemplo las conocidas cepas de tipo salvajeAppropriate strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum , are for example the known wild-type strains
Corynebacterium glutamicum ATCC13032 Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870 Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium melassecola ATCC17965 Corynebacterium melassecola ATCC17965
Brevibacterium flavum ATCC14067 Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 y Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020 Brevibacterium divaricatum ATCC14020
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
y mutantes o respectivamente cepas que producen L-lisina, preparadas a partir de ellas, tales como por ejemploand mutants or strains that produce L-lysine, prepared from them, such as for example
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Corynebacterium glutamicum FERM-P 1709 Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708 Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712 Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 Corynebacterium glutamicum FERM-P 6464
Corynebacterium glutamicum DSM5715 Corynebacterium glutamicum DSM5715
Corynebacterium glutamicum DSM12866 y Corynebacterium glutamicum DSM12866 and
Corynebacterium glutamicum DM58-1. Corynebacterium glutamicum DM58-1.
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Se hizo posible para los autores del invento aislar el nuevo gen dapC de C. glutamicum, que codifica la enzima N-succinil-aminocetopimelato transaminasa (EC 2.6.1.17).It became possible for the inventors to isolate the new dapC gene from C. glutamicum , which encodes the enzyme N-succinyl aminoacetopimelate transaminase (EC 2.6.1.17).
Para el aislamiento del gen dapC o también de otros genes de C. glutamicum, se establece en primer lugar un banco de genes de este microorganismo en el seno de E. coli. El establecimiento de bancos de genes ha sido descrito y redactado en libros de texto y manuales generalmente conocidos. Como ejemplo se han de mencionar el libro de texto de Winnacker: Gene und Klone. Eine Einführung in die Gentechnologie [Genes y clones. Una introducción en la tecnología génica] (editorial Verlag Chemie, Weinheim, Alemania, 1990) o el manual de Sambrook y colaboradores: Molecular Cloning, A Laboratory Manual Clonación molecular, un manual de laboratorio] (Cold Spring Harbor Laboratory Press, 1989). Un banco de genes muy conocido es el de la cepa W3110 de E. coli K-12, que fue establecido por Kohara y colaboradores (Cell 50, 495-508 (1987)) en el seno de vectores \lambda. Bathe y colaboradores (Molecular and General Genetics, 252:255-265, 1996) describen un banco de genes de C. glutamicum ATCC13032, que fue establecido con ayuda del vector cosmídico SuperCos I (Wahl y colaboradores 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) en el seno de la cepa NM554 de E. coli K-12 (Raleigh y colaboradores, 1988, Nucleic Acids Research 16:1563-1575). Börmann y colaboradores (Molecular Microbiology 6(3), 317-326 (1992)) a su vez describen un banco de genes de C. glutamicum ATCC13032 mediando utilización del cósmido pHC79 (Hohn y Collins, Gene 11, 291-298 (1980)).For the isolation of the dapC gene or also of other genes of C. glutamicum , a gene bank of this microorganism is first established within E. coli . The establishment of gene banks has been described and written in generally known textbooks and manuals. As an example, the textbook of Winnacker: Gene und Klone should be mentioned. Eine Einführung in die Gentechnologie [Genes and clones. An introduction in gene technology] (Verlag Chemie, Weinheim, Germany, 1990) or the Sambrook et al. Manual: Molecular Cloning, A Laboratory Manual Molecular cloning, a laboratory manual] (Cold Spring Harbor Laboratory Press, 1989). A well-known gene bank is that of E. coli K-12 strain W3110, which was established by Kohara et al. (Cell 50, 495-508 (1987)) within λ vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a gene bank of C. glutamicum ATCC13032, which was established with the help of the SuperCos I cosmetic vector (Wahl et al. 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164) in the E. coli strain K-12 NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Börmann et al. (Molecular Microbiology 6 (3), 317-326 (1992)) in turn describe a gene bank of C. glutamicum ATCC13032 mediating the use of the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980) ).
Para la producción de un banco de genes de C. glutamicum en el seno de E. coli se pueden utilizar también unos plásmidos tales como el pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) o el pUC9 (Vieira y colaboradores, 1982, Gene, 19:259-268). Como anfitrionas son adecuadas en especial aquellas cepas de E. coli que son defectuosas en cuanto a restricción y recombinación. Un ejemplo de esto es la cepa DH5\alphamcr, que fue descrita por Grant y colaboradores (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). Los fragmentos de ADN largos, clonados con ayuda de cósmidos, pueden ser subclonados a continuación a su vez en unos vectores corrientes, que son apropiados para la secuenciación, y a continuación pueden ser secuenciados, tal como se ha descrito p.ej. en la cita de Sanger y colaboradores (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).For the production of a gene bank of C. glutamicum within E. coli , plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira and collaborators, 1982, Gene, 19: 259-268). Especially suitable are those E. coli strains that are defective in terms of restriction and recombination. An example of this is strain DH5? Alphamcr, which was described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments, cloned with the aid of cosmids, can then be subcloned in turn into current vectors, which are appropriate for sequencing, and then can be sequenced, as described eg in the citation. from Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74: 5463-5467, 1977).
De este modo se obtuvo la nueva secuencia de ADN de C. glutamicum que codifica el gen dapC, que en la forma de SEQ ID NO 1 es una parte componente del presente invento. Además, a partir de la presente secuencia de ADN se puede deducir, con los métodos arriba descritos, la secuencia de aminoácidos de la correspondiente proteína. En SEQ ID NO 2 se representa la secuencia de aminoácidos que se establece, del producto génico de dapC.Thus, the new C. glutamicum DNA sequence encoding the dapC gene was obtained, which in the form of SEQ ID NO 1 is a component part of the present invention. Furthermore, from the present DNA sequence, the amino acid sequence of the corresponding protein can be deduced with the methods described above. SEQ ID NO 2 represents the amino acid sequence that is established, of the dapC gene product.
Una secuencias de ADN codificadoras, que se establecen a partir de SEQ ID NO 1 mediante la degenerabilidad del código genético, son asimismo una parte componente del invento. De igual modo, son parte componente del invento unas secuencias de ADN, que se hibridan con SEQ ID NO 1 o con partes de SEQ ID NO 1. En el mundo especializado se conocen además unos intercambios conservativos de aminoácidos, tales como p.ej. un intercambio de glicina por alanina o de ácido aspártico por ácido glutámico en proteínas como "mutaciones de sentido" (en inglés sense mutations), que no conducen a ninguna modificación fundamental de la actividad de la proteína, es decir son funcionalmente neutras. Además, es conocido que unas modificaciones en el extremo terminal de N y/o de C de una proteína, cuya función no perjudican esencialmente o incluso pueden estabilizar. Datos acerca de esto los encuentra un especialista en la materia, entre otras citas, en las de Ben-Bassat y colaboradores (Journal of Bacteriology 169:751-757 (1987)), de O'Regan y colaboradores (Gene 77:237-251 (1989)), de Sahin-Toth y colaboradores (Protein Sciences 3:240-247 (1994)), y de Hochuli y colaboradores (Bio/Technology 6:1321-1325 (1988)) y en libros de texto conocidos acerca de la genética y la biología molecular. Unas secuencias de aminoácidos, que se establecen de una manera correspondiente a partir de SEQ ID NO 2, son asimismo una parte componente del invento.A coding DNA sequences, which are established from SEQ ID NO 1 through the degenerability of the genetic code, are also a component part of the invention. From likewise, sequences of DNA, which hybridize with SEQ ID NO 1 or with parts of SEQ ID NO 1. In the specialized world also knows some exchanges amino acid preservatives, such as eg an exchange of alanine glycine or aspartic acid by glutamic acid in proteins like "sense mutations" mutations), which do not lead to any fundamental modification of the protein activity, that is to say they are functionally neutral. In addition, it is known that some modifications at the terminal end of N and / or C of a protein, whose function does not impair essentially or can even stabilize. Facts about this they are found by a specialist in the field, among other appointments, in those of Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), by O'Regan and collaborators (Gene 77: 237-251 (1989)), of Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)), and de Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and in books of Known text about genetics and molecular biology. Nail amino acid sequences, which are established in a way corresponding from SEQ ID NO 2, they are also a part component of the invention.
De igual manera, son una parte componente del invento unas secuencias de ADN, que se hibridan con la SEQ ID NO 1 o con partes de la SEQ ID NO 1. Finalmente, son una parte componente del invento unas secuencias de ADN, que se producen mediante la reacción en cadena de la polimerasa (PCR) con utilización de unos cebadores, que se establecen a partir de SEQ ID NO 1. Tales oligonucleótidos tienen típicamente una longitud de por lo menos 15 nucleótidos.Similarly, they are a component part of I invent DNA sequences, which hybridize with SEQ ID NO 1 or with parts of SEQ ID NO 1. Finally, they are a component part of the invention DNA sequences, which are produced by the polymerase chain reaction (PCR) using some primers, which are established from SEQ ID NO 1. Such oligonucleotides typically have a length of at least 15 nucleotides
Unas instrucciones para la identificación de secuencias de ADN mediante hibridación, las encuentra un especialista en la materia, entre otras citas, en el manual "The DIG System Users Guide for Filter Hybridization" [La guía para los usuarios del sistema DIG] de la entidad Roche Diagnostics GmbH (Mannheim, Alemania, 1993) y en la cita de Liebl y colaboradores (International Journal of Systematic Bacteriology (1991) 41: 255-260). Unas instrucciones para la amplificación de secuencias de ADN con ayuda de la reacción en cadena de la polimerasa (PCR) las encuentra un especialista en la materia, entre otras citas, en el manual de Gait: Oligonucleotide Synthesis: A Practical Approach [Síntesis de oligonucleótidos: Un enfoque práctico] (IRL Press, Oxford, Reino Unido, 1984) y en la cita de Newton y Graham: PCR (editorial Spektrum Akademischer Verlag, Heidelberg, Alemania, 1994).Instructions for the identification of DNA sequences by hybridization, found by a specialist in the field, among other quotes, in the manual "The DIG System Users Guide for Filter Hybridization "[The guide for DIG system users] of the Roche Diagnostics GmbH (Mannheim, Germany, 1993) and at the appointment of Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). Some instructions for amplification of DNA sequences with the help of the chain reaction of the Polymerase (PCR) is found by a specialist in the field, among other quotes, in the Gait manual: Oligonucleotide Synthesis: A Practical Approach [Oligonucleotide Synthesis: An Approach practical] (IRL Press, Oxford, United Kingdom, 1984) and at the appointment of Newton and Graham: PCR (editorial Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
Los autores del invento descubrieron que unas bacterias corineformes, después de una sobreexpresión del gen dapC, producen L-lisina de una manera mejorada.The authors of the invention discovered that some Coryneform bacteria, after overexpression of the dapC gene, They produce L-lysine in an improved way.
Para la consecución de una sobreexpresión, se puede aumentar el número de copias de los correspondientes genes, o se puede mutar la región de promotor y de regulación o el sitio de fijación a ribosomas, que se encuentra secuencia arriba del gen estructural. De igual manera, actúan unas casetes de expresión, que son incorporadas secuencia arriba del gen estructural. Mediante unos promotores inducibles es posible adicionalmente aumentar la expresión en el transcurso de la producción por fermentación de L-lisina. Mediante unas medidas técnicas para la prolongación de la duración de vida del ARNm (ácido ribonucleico mensajero) se mejora asimismo la expresión. Además, mediante evitación de la degradación de la proteína enzimática, se refuerza asimismo la actividad enzimática. Los genes o las construcciones artificiales de genes, o bien pueden presentarse en plásmidos con un diverso número de copias o pueden ser integrados y amplificados en el cromosoma. Alternativamente, además se puede conseguir una sobreexpresión de los correspondientes genes mediante una modificación de la composición del medio y de la realización del cultivo.To achieve overexpression, it can increase the number of copies of the corresponding genes, or the promoter and regulatory region or the site of ribosome binding, which is found upstream of the gene structural. Similarly, expression cassettes act, which They are incorporated upstream of the structural gene. Through inducible promoters it is possible to further increase the expression in the course of fermentation production of L-lysine Through technical measures for prolongation of the life span of mRNA (ribonucleic acid messenger) expression is also improved. In addition, by Avoidance of degradation of enzymatic protein, is reinforced also enzymatic activity. The genes or the constructions artificial genes, or they can occur in plasmids with a different number of copies or can be integrated and amplified on the chromosome Alternatively, you can also get a overexpression of the corresponding genes through a modification of the composition of the medium and the realization of culture.
Unas instrucciones acerca de esto las encuentra el especialista en la materia, entre otras citas, en las de Martin y colaboradores (Bio/Technology 5, 137-146 (1987)), de Guerrero y colaboradores (Gene 138, 35-41 (1994)), de Tsuchiya y Morinaga (Bio/Technology 6, 428-430 (1988)), de Eikmanns y colaboradores (Gene 102, 93-98 (1991)), del documento de patente europea EP 0 472 869, del documento de patente de los EE.UU. US 4.601.893, de Schwarzer y Pühler (Bio/Technology 9, 84-87 (1991), de Reinscheid y colaboradores (Applied and Environmental Microbiology 60, 126-132 (1994)), de LaBarre y colaboradores (Journal of Bacteriology 175, 1001-1007 (1993)), del documento de solicitud de patente internacional WO 96/15246, de Malumbres y colaboradores (Gene 134, 15-24 (1993)), del documento de solicitud de patente japonesa JP-A-10-229891, de Jensen y Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), de Makrides (Microbiological Reviews 60:512-538 (1996)) y en libros de texto conocidos acerca de la genética y la biología molecular.Some instructions about this are found the specialist in the field, among other appointments, in Martin's and collaborators (Bio / Technology 5, 137-146 (1987)), de Guerrero et al. (Gene 138, 35-41 (1994)), by Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), by Eikmanns et al. (Gene 102, 93-98 (1991)), of the patent document European EP 0 472 869, from US Pat. US 4,601,893, by Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991), by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), from LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), of the application document for International Patent WO 96/15246, of Malitudes and collaborators (Gene 134, 15-24 (1993)), of the document of Japanese patent application JP-A-10-229891, of Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), by Makrides (Microbiological Reviews 60: 512-538 (1996)) and in textbooks known about genetics and molecular biology.
A modo de ejemplo, el gen dapC conforme al invento fue sobreexpresado con ayuda de plásmidos.As an example, the dapC gene according to invention was overexpressed with the help of plasmids.
Como plásmidos son apropiados los que se replican en bacterias corineformes. Numerosos vectores plasmídicos conocidos, tales como p.ej. el pZ1 (Menkel y colaboradores, Applied and Environmental Microbiology (1989) 64: 549-554), el pEKEx1 (Eikmanns y colaboradores, Gene 102:93-98 (1991)) o el pHS2-1 (Sonnen y colaboradores, Gene 107:69-74 (1991)) se basan en los plásmidos crípticos pHM1519, pBL1 o pGA1. Otros vectores plasmídicos tales como p.ej. los que se basan en el pCG4 (documento US-A 4.489.160), o el pNG2 (Serwold-Davis y colaboradores, FEMS Microbiology Letters 66, 119-124 (1990)), o el pAG1 (documento US-A 5.158.891) se pueden utilizar de igual manera.Suitable plasmids are those that are replicate in coryneform bacteria. Numerous plasmid vectors known, such as e.g. pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), the pEKEx1 (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on plasmids cryptic pHM1519, pBL1 or pGA1. Other such plasmid vectors such as those based on pCG4 (document US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (document US-A 5,158,891) can be used equally way.
Un ejemplo de un plásmido, con cuya ayuda se puede sobreexpresar el gen dapC, es el vector de lanzadera (en inglés shuttle vector) de E. coli - C. glutamicum pXT-dapCexp. El contiene la región de replicación rep del plásmido pGA1, incluyendo el efector de replicación per (documento US-A-5.175.108; Nesvera y colaboradores, Journal of Bacteriology 179, 1525-1532 (1997)), el gen tetA(Z) del plásmido pAG1 que media en la resistencia a tetraciclina (documento US-A-5.158.891; inscripción en el banco de genes en el National Center for Biotechnology Information (NCBI, Bethesda, MD, EE.UU.) con el número de acceso AF121000), así como el origen de replicación, el promotor trc, las regiones de terminación T1 y T2 y el gen lacl^{q} (represor del operón lac de E. coli) del plásmido pTRC99A (Amann y colaboradores (1988), Gene 69: 301-315).An example of a plasmid, with whose help the dapC gene can be overexpressed, is the shuttle vector (in English shuttle vector) of E. coli - C. glutamicum pXT-dapCexp. It contains the rep replication region of plasmid pGA1, including the per-replication effector (US-A-5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetA (Z) gene of plasmid pAG1 mediating tetracycline resistance (US-A-5,158,891; enrollment in the gene bank at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with the accession number AF121000), as well as the origin of replication, the trc promoter, the T1 and T2 termination regions and the laclq gene (repressor of the E. coli lac operon) of plasmid pTRC99A (Amann et al. (1988), Gene 69: 301-315).
El vector de lanzadera pXT-dapCexp se representa en la Figura 2.The shuttle vector pXT-dapCexp is represented in Figure 2.
Además son apropiados aquellos vectores plasmídicos, con cuya ayuda se puede aplicar el procedimiento de la amplificación de genes por integración en el cromosoma, tal como fue descrito por ejemplo por Reinscheid y colaboradores (Applied and Environmental Microbiology 60, 126-132 (1994)) para la duplicación o respectivamente amplificación del operón hom-thrB. En el caso de este método, el gen completo es clonado en un vector plasmídico, que puede replicar en un anfitrión (típicamente E. coli), pero no en C. glutamicum. Como vectores entran en consideración por ejemplo el pSUP301 (Simon y colaboradores, Bio/Technology 1, 784-791 (1983)), el pK18mob o pK19mob (Schäfer y colaboradores, Gene 145, 69-73 (1994)), el pGEM-T (Promega corporation, Madison, WI, EE.UU.), el pCR2.1-TOPO (Shuman (11994). Journal of Biological Chemistry 269:32678-84; documento US-A 5,487,993), el pCR®Blunt (Firma Invitrogen, Groningen, Holanda; Bernard y colaboradores, Journal of Molecular Biology, 234: 534-541 (1993)) o el pEM1 (Schrumpf y colaboradores, 1991, Journal of Bacteriology 173:4510-4516). El vector plasmídico que contiene el gen que se ha de amplificar, es transformado a continuación por conjugación o transformación en el seno de la respectiva cepa de C. glutamicum. El método de la conjugación ha sido descrito por ejemplo en la cita de Schäfer y colaboradores (Applied and Environmental Microbiology 60, 756-759 (1999)). Unos métodos para la transformación se han descrito por ejemplo en las citas de Thierbach y colaboradores (Applied Microbiology and Biotechnology 29, 356-362 (1988)), de Dunican y Shivnan (Bio/Technology 7, 1067-1070 (1989)) y de Tauch y colaboradores (FEMS Microbiological Letters 123, 343-347 (1994)). Después de una recombinación homóloga mediante un suceso de cruce [en inglés "cross over"], la cepa resultante contiene por lo menos dos copias del correspondiente gen.Also suitable are those plasmid vectors, with whose help the gene amplification procedure can be applied by integration into the chromosome, as described for example by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or respectively amplification of the hom-thrB operon. In the case of this method, the entire gene is cloned into a plasmid vector, which can replicate in a host (typically E. coli ), but not in C. glutamicum . Vectors include, for example, pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM- T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (11994). Journal of Biological Chemistry 269: 32678-84; US-A 5,487,993), pCR®Blunt (Signature Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516). The plasmid vector containing the gene to be amplified is then transformed by conjugation or transformation within the respective strain of C. glutamicum . The method of conjugation has been described for example in the quotation of Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1999)). Methods for transformation have been described, for example, in the quotations of Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and by Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination through a cross-over event, the resulting strain contains at least two copies of the corresponding gene.
Además, se encontró que mediante intercambio del aminoácido L-prolina en la posición 209 de la proteína enzimática N-succinil-aminocetopimelato transaminasa (véase SEQ ID No. 2) por cualquier otro aminoácido proteinógeno, en particular L-leucina (véase SEQ ID No. 4), con excepción de la L-prolina, se inicia un refuerzo, y unas bacterias corineformes, que llevan el correspondiente intercambio de aminoácidos, producen L-lisina de una manera mejorada. El intercambio de L-prolina por L-leucina en la posición 209 de la secuencia de aminoácidos puede efectuarse preferiblemente mediante el intercambio de la nucleobase citosina situada en la posición 716 por timina, tal como se representa en la secuencia de nucleótidos según SEQ ID No. 3.In addition, it was found that by exchange of amino acid L-proline at position 209 of the enzymatic protein N-succinyl aminoacetopimelate transaminase (see SEQ ID No. 2) for any other amino acid proteinogen, in particular L-leucine (see SEQ ID No. 4), with the exception of L-proline, a reinforcement, and coryneform bacteria, which carry the corresponding amino acid exchange, produce L-lysine in an improved way. The exchange of L-proline by L-leucine in the position 209 of the amino acid sequence can be effected preferably by exchanging the cytosine nucleobase located at position 716 by thymine, as depicted in the nucleotide sequence according to SEQ ID No. 3.
Para la mutagénesis se pueden utilizar unos procedimientos clásicos de mutagénesis mediando utilización de sustancias mutágenas tales como, por ejemplo, la N-metil-N'-nitro-N-nitroso-guanidina o una luz ultravioleta. Además, para la mutagénesis se pueden utilizar unos métodos in vitro, tales como por ejemplo un tratamiento con hidroxilamina (Molecular and General Genetics 145, 101 pp (1978)) o bien el uso der oligonucleótidos mutágenos (T. A. Brown: Gentechnologie für Einsteiger [Tecnología genética para novatos], Spektrum Akademischer Verlag, Heidelberg, 1993) o la reacción en cadena de la polimerasa (PCR), tal como se ha descrito en el manual de Newton y Graham (PCR, Spektrum Akademischer Verlag, Heidelberg, 1994).For mutagenesis, classical mutagenesis procedures can be used mediating the use of mutagenic substances such as, for example, N-methyl-N'-nitro-N-nitroso-guanidine or ultraviolet light. In addition, in vitro methods can be used for mutagenesis, such as for example a hydroxylamine treatment (Molecular and General Genetics 145, 101 pp (1978)) or the use of mutagenic oligonucleotides (TA Brown: Gentechnologie für Einsteiger [Technology genetics for newbies], Spektrum Akademischer Verlag, Heidelberg, 1993) or polymerase chain reaction (PCR), as described in the Newton and Graham manual (PCR, Spektrum Akademischer Verlag, Heidelberg, 1994).
Son objeto del invento de una manera correspondiente, además, unas bacterias corineformes que contienen la proteína enzimática N-succinil-aminocetopimelato transaminasa, en las cuales la secuencia de aminoácidos mostrada dentro de SEQ ID No. 2 ha sido intercambiada en la posición 209 por otro aminoácido distinto, exceptuando la L-prolina. Un aspecto adicional de este invento son unas bacterias corineformes, que contienen una correspondiente proteína enzimática, en la que el aminoácido L-prolina ha sido intercambiado en la posición 209 de la proteína enzimática (véase SEQ ID No. 2) por L-leucina (véase SEQ ID No. 4).They are object of the invention in a way corresponding, in addition, coryneform bacteria that contain enzymatic protein N-succinyl aminoacetopimelate transaminase, in which the amino acid sequence shown within SEQ ID No. 2 has been exchanged at position 209 for another different amino acid, except for L-proline. An additional aspect of this invention are bacteria coryneforms, which contain a corresponding protein enzymatic, in which the amino acid L-proline has been exchanged at position 209 of the enzymatic protein (see SEQ ID No. 2) for L-leucine (see SEQ ID No. 4).
Un objeto adicional del invento son, de una manera correspondiente, unas secuencias de polinucleótidos que proceden de bacterias corineformes, las cuales contienen unos genes que codifican las mencionadas proteínas enzimáticas N-succinil-aminocetopimelato transaminasas.A further object of the invention are, of a correspondingly, polynucleotide sequences that they come from coryneform bacteria, which contain genes encoding the aforementioned enzymatic proteins N-succinyl aminoacetopimelate transaminases
Todavía un objeto de este invento son unas bacterias corineformes, que contienen un ADN que codifica una proteína enzimática N-succinil-aminocetopimelato transaminasa, que está caracterizada por el intercambio de L-prolina por L-leucina en la posición 209, cuyo ADN, en lugar de la nucleobase citosina en la posición 716 (véase SEQ ID No. 1) contiene timina, tal como se representa en SEQ ID No. 3.Still an object of this invention are some coryneform bacteria, which contain a DNA that encodes a enzymatic protein N-succinyl aminoacetopimelate transaminase, which is characterized by the exchange of L-proline by L-leucine in the position 209, whose DNA, instead of the cytosine nucleobase in the position 716 (see SEQ ID No. 1) contains thymine, as Represents in SEQ ID No. 3.
Adicionalmente, puede ser ventajoso para la producción de L-lisina reforzar o sobreexpresar, junto al gen dapC, una o varias enzimas de la correspondiente ruta de biosíntesis, de la glicolisis, de la anaplerótica o de la exportación de aminoácidos.Additionally, it can be advantageous for the L-lysine production reinforce or overexpress, next to the dapC gene, one or several enzymes of the corresponding route of biosynthesis, glycolysis, anaplerotic or amino acid export
Así, por ejemplo, adicionalmente al gen dapC, se puede(n) sobreexpresar o amplificar al mismo tiempo uno o varios de los genes seleccionados entre el conjunto formado porThus, for example, in addition to the dapC gene, can overexpress or amplify at the same time one or several of the genes selected from the set consisting of
- \bullet?
- el gen lysC que codifica una aspartato cinasa resistente a la retroalimentación (feed back) (Kalinowski y colaboradores (1990), Molecular and General Genetics 224: 317-324), the lysC gene that encodes a aspartate kinase resistant to feedback (feed back) (Kalinowski et al. (1990), Molecular and General Genetics 224: 317-324),
- \bullet?
- el gen asd que codifica la aspartato-semialdehído deshidrogenasa (documento EP-A 0 219 027; Kalinowski y colaboradores (1991), Molecular Microbiology 5:1197-1204, y Kalinowski y colaboradores (1991), Molecular and General Genetics 224: 317-324), the asd gene that encodes the aspartate-semialdehyde dehydrogenase (document EP-A 0 219 027; Kalinowski et al. (1991), Molecular Microbiology 5: 1197-1204, and Kalinowski and collaborators (1991), Molecular and General Genetics 224: 317-324),
\global\parskip1.000000\baselineskip\ global \ parskip1.000000 \ baselineskip
- \bullet?
- el gen dapA que codifica la dihidrodipicolinato sintasa (documento EP-B 0 197 335), the dapA gene that encodes the dihydrodipicolinate synthase (EP-B 0 197 335),
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- el gen dapB que codifica la dihidrodipicolinato reductasa (inscripción en el banco de genes número de acceso X67737; Pisabarro y colaboradores (1993), Journal of Bacteriology, 175(9): 2743-2749), the dapB gene that encodes the dihydrodipicolinate reductase (registration in the gene bank access number X67737; Pisabarro et al. (1993), Journal of Bacteriology, 175 (9): 2743-2749),
- \bullet?
- el gen dapD que codifica la tetrahidro-picolinato succinilasa (inscripción en el banco de genes número de acceso AJ004934; Wehrmann y colaboradores (1998), Journal of Bacteriology 180: 3159-3163), the dapD gene that encodes the tetrahydro-picolinate succinylase (inscription in the gene bank access number AJ004934; Wehrmann et al. (1998), Journal of Bacteriology 180: 3159-3163),
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- el gen dapE que codifica la N-succinil-diaminopimelato dessuccinilasa (inscripción en el banco de genes número de acceso X81379; Wehrmann y colaboradores (1994), Microbiology 140: 3349-3356), the dapE gene that encodes the N-succinyl diaminopimelate dessuccinilase (registration in the gene bank access number X81379; Wehrmann et al. (1994), Microbiology 140: 3349-3356),
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- el gen dapF que codifica la diaminopimelato epimerasa (documento DE: 199 43 587.1, DSM12968), the dapF gene that encodes the diaminopimelate epimerase (DE document: 199 43 587.1, DSM12968),
- \bullet?
- el gen lysA que codifica la diaminopimelato descarboxilasa (inscripción en el banco de genes número de acceso X07563; Yeh y colaboradores (1988), Molecular and General Genetics 212: 112-119), the lysA gene that encodes the diaminopimelate decarboxylase (registration in the gene bank access number X07563; Yeh et al. (1988), Molecular and General Genetics 212: 112-119),
- \bullet?
- el gen ddh que codifica la diaminopimelato deshidrogenasa (Ishino y colaboradores (1988), Agricultural and Biological Chemistry 52(11): 2903-2909), the ddh gene that encodes the diaminopimelate dehydrogenase (Ishino et al. (1988), Agricultural and Biological Chemistry 52 (11): 2903-2909),
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- el gen lysE que codifica la exportación de lisina (documento DE-A-195 48 222), the lysE gene that encodes the lysine export (document DE-A-195 48 222),
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- el gen pyc que codifica la piruvato carboxilasa (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), the pyc gene that encodes the pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
\bullet el gen mqo que codifica la malato-quinona oxidorreductasa (Molenaar y colaboradores (1998), European Journal of Biochemistry 254: 395-403),the mqo gene that encodes the Malate-quinone oxidoreductase (Molenaar and collaborators (1998), European Journal of Biochemistry 254: 395-403),
\bullet el gen zwa1 (documento DE: 19959328.0, DSM 13115)The zwa1 gene (DE document: 19959328.0, DSM 13115)
\bullet el gen gdh que codifica la glutamato deshidrogenasa (Börmann y colaboradores (1992), Molecular Microbiology 6, 317-326). Se prefiere el refuerzo simultáneo de uno o varios genes, seleccionados entre el conjunto formado por dapD, dapE y dapF.? the gdh gene encoding glutamate dehydrogenase (Börmann et al. (1992), Molecular Microbiology 6, 317-326). Reinforcement is preferred simultaneous one or more genes, selected from the set formed by dapD, dapE and dapF.
Además, para la producción de L-lisina puede ser ventajoso, de modo adicional al refuerzo del gen dapC, eventualmente en combinación con uno o varios genes seleccionados entre el conjunto formado por dapD, dapE y dapF, debilitar al mismo tiempoIn addition, for the production of L-lysine can be advantageous, in addition to the dapC gene reinforcement, possibly in combination with one or more genes selected from the set consisting of dapD, dapE and dapF, weaken at the same time
- \bullet?
- al gen pck que codifica la fosfoenolpiruvato carboxicinasa (documento DE 199 50 409.1, DSM 13047), o to the pck gene that encodes the phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047), or
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- al gen pgi que codifica la glucosa-6-fosfato isomerasa (documento US 09/396,478, DSM 12969), o to the pgi gene that encodes the glucose-6-phosphate isomerase (US 09 / 396,478, DSM 12969), or
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- al gen poxB que codifica la piruvato oxidasa (documento DE: 19951975.7, DSM 13114), o to the poxB gene that encodes the pyruvate oxidase (DE: 19951975.7, DSM 13114), or
- \bullet?
- al gen zwa2(documento DE: 19959327.2, DSM 13113), oto the zwa2 gene (DE document: 19959327.2, DSM 13113), or
- \bullet?
- al gen sucC o sucD que codifica la succinil-CoA sintetasa (documento DE: 19956686.0) to the sucC or sucD gene that encodes succinyl-CoA synthetase (DE document: 19956686.0)
Además, para la producción de L-lisina puede ser ventajoso, de modo adicional al refuerzo del gen dapC, eventualmente en combinación con uno o varios genes seleccionados entre el conjunto formado por dapD, dapE y dapF, excluir reacciones secundarias indeseadas (Nakayama: "Breeding of Amino Acid Producing Micro-organisms" [Cría de microorganismos productores de aminoácidos], in: Overproduction of Microbial Products [Sobreproducción de productos microbianos], Krumphanzl, Sikyta, Vanek (coordinadores de edición), Academic Press, Londres, Reino Unido, 1982).In addition, for the production of L-lysine can be advantageous, in addition to the dapC gene reinforcement, possibly in combination with one or several genes selected from the set consisting of dapD, dapE and dapF, exclude unwanted side reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms "[Breeding of microorganisms amino acid producers], in: Overproduction of Microbial Products [Overproduction of microbial products], Krumphanzl, Sikyta, Vanek (editing coordinators), Academic Press, London, United Kingdom, 1982).
Los microorganismos producidos conforme al invento pueden ser cultivados de un modo continuo o discontinuo según el procedimiento batch (cultivación por tandas) o según el procedimiento fed batch (procedimiento de afluencia) o según el procedimiento fed batch repeated (procedimiento repetitivo de afluencia) con la finalidad de efectuar la producción de L-lisina. Una recopilación acerca de conocidos métodos de cultivación se describe en el libro de texto de Chmiel (Bioprozeßtechnik 1. Einführung in die Bioverfahrenstechnik [Técnica de los bioprocesos 1. Introducción en la técnica de los procedimientos biológicos] (editorial Gustav Fischer, Stuttgart, 1991)) o en el libro de texto de Storhas (Bioreaktoren und periphere Einrichtungen [Biorreactores y disposiciones periféricas] (editorial Vieweg, Braunschweig/Wiesbaden, 1999)).The microorganisms produced according to invention can be grown continuously or discontinuously according to the batch procedure (batch cultivation) or according to the fed batch procedure (inflow procedure) or according to fed batch repeated procedure influx) in order to effect the production of L-lysine A compilation about acquaintances Cultivation methods described in Chmiel's textbook (Bioprozeßtechnik 1. Einführung in die Bioverfahrenstechnik [Technique of bioprocesses 1. Introduction to the technique of biological procedures] (editorial Gustav Fischer, Stuttgart, 1991)) or in the textbook of Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral arrangements] (Vieweg editorial, Braunschweig / Wiesbaden, 1999).
El medio de cultivo que se ha de utilizar debe de satisfacer de una manera apropiada las exigencias de las respectivas cepas. Unas descripciones de medios de cultivo de diferentes microorganismos están contenidas en el manual "Manual of Methods for General Bacteriology" [Manual de métodos para la bacteriología general] de la American Society for Bacteriology (Washington D.C., EE.UU., 1981).The culture medium to be used must to properly meet the demands of the respective strains. A description of culture media of different microorganisms are contained in the manual "Manual of Methods for General Bacteriology " general bacteriology] of the American Society for Bacteriology (Washington D.C., USA, 1981).
Como fuente de carbono se pueden utilizar azúcares e hidratos de carbono tales como p.ej. glucosa, sacarosa, lactosa, fructosa, melazas, almidones y celulosas, aceites y grasas tales como p.ej. aceite de soja, aceite de girasol, aceite de cacahuete y grasa de coco, ácidos grasos tales como p.ej. ácido palmítico, ácido esteárico y ácido linoleico, alcoholes tales como p.ej. glicerol y etanol, y ácidos orgánicos tales como p.ej. ácido acético. Estas sustancias se pueden utilizar individualmente o como una mezcla.As carbon source can be used sugars and carbohydrates such as eg glucose, sucrose, lactose, fructose, molasses, starches and cellulose, oils and fats such as eg soybean oil, sunflower oil, peanut and coconut fat, fatty acids such as eg acid palmitic, stearic acid and linoleic acid, alcohols such as eg glycerol and ethanol, and organic acids such as eg acid acetic. These substances can be used individually or as a mix.
Como fuente de nitrógeno se pueden utilizar compuestos orgánicos que contienen nitrógeno, tales como peptonas, un extracto de levadura, un extracto de carne, un extracto de malta, un líquido de maceración de maíz, harina de soja y urea, o compuestos inorgánicos, tales como sulfato de amonio, cloruro de amonio, fosfato de amonio, carbonato de amonio y nitrato de amonio. Las fuentes de nitrógeno se pueden utilizar individualmente o en forma de una mezcla.As a source of nitrogen they can be used organic compounds that contain nitrogen, such as peptones, a yeast extract, a meat extract, a malt extract, a maceration liquid of corn, soybean meal and urea, or inorganic compounds, such as ammonium sulfate, chloride ammonium, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can be used individually or in form of a mixture.
Como fuente de fósforo se pueden utilizar ácido fosfórico, dihidrógeno-fosfato de potasio o hidrógeno-fosfato de dipotasio o las correspondientes sales que contienen sodio. El medio de cultivo debe de contener además ciertas sales de metales tales como p.ej. sulfato de magnesio o sulfato de hierro, que son necesarias para el crecimiento. Finalmente, de una manera adicional a las sustancias arriba mencionadas, se pueden emplear hormonas esenciales tales como aminoácidos y vitaminas. Al medio de cultivo se le pueden añadir, además de esto, unos compuestos precursores apropiados. Las mencionadas sustancias empleadas se pueden añadir al cultivo en forma de una tanda única o se pueden aportar como alimentación de una manera apropiada durante la cultivación.As a source of phosphorus acid can be used phosphoric, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding salts containing sodium. The culture medium must also contain certain metal salts such as e.g. magnesium sulfate or iron sulfate, which are necessary for the increase. Finally, in an additional way to substances above mentioned, essential hormones such as like amino acids and vitamins. The culture medium can be add, in addition to this, appropriate precursor compounds. The mentioned substances used can be added to the crop in shape of a single batch or can be provided as food for an appropriate way during cultivation.
Para el control del pH del cultivo, se emplean de manera apropiada ciertos compuestos de carácter básico, tales como hidróxido de sodio, hidróxido de potasio, amoníaco o respectivamente agua amoniacal, o ciertos compuestos de carácter ácido, tales como ácido fosfórico o ácido sulfúrico. Para la represión del desarrollo de espuma se pueden emplear agentes antiespumantes tales como p.ej. ésteres poliglicólicos de ácidos grasos. Para la conservación de la estabilidad de los plásmidos, se pueden añadir al medio unas apropiadas sustancias que actúan selectivamente, tales como p.ej. antibióticos. Con el fin de mantener unas condiciones aerobias, se incorporan en el cultivo oxígeno o mezclas gaseosas que contienen oxígeno, tales como p.ej. aire. La temperatura del cultivo está situada normalmente en 20ºC hasta 45ºC y de manera preferida en 25ºC hasta 40ºC. El proceso de cultivo se prosigue durante tanto tiempo hasta que se haya formado un máximo de lisina. Esta meta se alcanza normalmente en el transcurso de 10 horas hasta 160 horas.For the control of the pH of the culture, they are used appropriately certain basic compounds, such such as sodium hydroxide, potassium hydroxide, ammonia or respectively ammonia water, or certain compounds of character acid, such as phosphoric acid or sulfuric acid. For the repression of foam development agents can be used defoamers such as eg polyglycolic acid esters fatty For the conservation of plasmid stability, it is they can add appropriate substances that act selectively, such as antibiotics. With the purpose of maintain aerobic conditions, are incorporated into the crop oxygen or gaseous mixtures containing oxygen, such as e.g. air. The culture temperature is normally located at 20 ° C up to 45 ° C and preferably at 25 ° C to 40 ° C. The process of cultivation is continued for so long until it has formed A maximum of lysine. This goal is normally achieved in the 10 hours to 160 hours.
El análisis de L-lisina se puede efectuar mediante una cromatografía de intercambio de aniones con una subsiguiente derivatización con ninhidrina, tal como se describe en la cita de Spackman y colaboradores (Analytical Chemistry, 30, (1958), 1190).L-lysine analysis can be perform by an anion exchange chromatography with a subsequent derivatization with ninhydrin, as described in the appointment of Spackman et al. (Analytical Chemistry, 30, (1958), 1190).
El siguiente microorganismo fue depositado en la Deutsche Sammlung für Mikrorganismen und Zellkulturen (DSMZ = Colección Alemana de Microorganismos y Cultivos Celulares], Braunschweig, Alemania) conforme al Convenio de Budapest:The following microorganism was deposited in the Deutsche Sammlung für Mikrorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures], Braunschweig, Germany) under the Budapest Convention:
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- Corynebacterium glutamicum cepa DSM5715/pXT-dapCexp como DSM 13254; Corynebacterium glutamicum strain DSM5715 / pXT-dapCexp as DSM 13254;
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- Corynebacterium glutamicum cepa DSM5715/pJC1dapF como DSM 13382; Corynebacterium glutamicum strain DSM5715 / pJC1dapF as DSM 13382;
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- Corynebacterium glutamicum cepa DSM5715/pJC1dapDE como DSM 13383; Corynebacterium glutamicum strain DSM5715 / pJC1dapDE as DSM 13383;
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- Corynebacterium glutamicum cepa DSM5715/pJC1dapDEF como DSM 13384. Corynebacterium glutamicum strain DSM5715 / pJC1dapDEF as DSM 13384.
El procedimiento conforme al invento sirve para la preparación de L-lisina por fermentación.The process according to the invention serves to the preparation of L-lysine by fermentation.
El presente invento es explicado con mayor detalle a continuación con ayuda de Ejemplos de realización.The present invention is explained in greater detail. detail below with the help of Examples of realization.
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Un ADN cromosomal procedente de Corynebacterium glutamicum ATCC 13032 fue aislado tal como se describe en la cita de Tauch y colaboradores (1995, Plasmid 33:168-179) y disociado parcialmente con la enzima de restricción Sau3AI (Amersham Pharmacia, Freiburg, Alemania, Descripción del producto Sau3AI, Nº de código 27-0913-02). Los fragmentos de ADN fueron desfosforilados con una fosfatasa alcalina de camarón (Roche Diagnostics GmbH, Mannheim, Alemania, Descripción del producto SAP, Nº de código 1758250). El ADN del vector cosmídico SuperCos1 (Wahl y colaboradores (1987) Proceedings of the National Academy of Sciences EE.UU. 84:2160-2164) adquirido de la entidad Stratagene (La Jolla, EE.UU., Descripción del producto SuperCos1 Cosmid Vektor Kit, Nº de código 251301), fue disociado con la enzima de restricción XbaI (Amersham Pharmacia, Freiburg, Alemania, Descripción del producto XbaI, Nº de código 27-0948-02) y asimismo desfosforilado con una fosfatasa alcalina de camarón. A continuación, el ADN de cósmido se disoció con la enzima de restricción BamHI (Amersham Pharmacia, Freiburg, Alemania, Descripción del producto BamHI, Nº de código 27-0868-04). El ADN de cósmido, tratado de esta manera, se mezcló con el ADN de ATCC 13032 tratado, y la tanda se trató con la ligasa de ADN de T4 (Amersham Pharmacia, Freiburg, Alemania, Descripción del producto T4-DNA-Ligase, Nº de código 27-0870-04). La mezcla de ligacion fue a continuación empaquetada en fagos con ayuda del Extracto de Empaquetamiento Gigapack II XL (Stratagene, La Jolla, EE.UU., Descripción del producto Gigapack II XL Packing Extract, Nº de código 200217). Para la infección de la cepa NM554 de E. coli (Raleigh y colaboradores 1988, Nucleic Acid Research 16:1563-1575) las células se recogieron en MgSO_{4} 10 mM y se mezclaron con una parte alícuota de la suspensión de fagos. La infección y la titulación del banco de cósmidos se llevaron a cabo tal como se describe en la cita de Sambrook y colaboradores (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), habiendo sido sembradas en placas las células sobre un agar LB (Lennox, 1955, Virology, 1:190) con 100 mg/l de ampicilina. Después de una incubación durante una noche a 37ºC se seleccionaron clones individuales recombinantes.A chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described in the quote by Tauch et al. (1995, Plasmid 33: 168-179) and partially dissociated with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Description of the product Sau3AI, code number 27-0913-02). The DNA fragments were dephosphorylated with an alkaline shrimp phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, SAP Product Description, Code No. 1758250). The SuperCos1 cosmid vector DNA (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164) acquired from the Stratagene entity (La Jolla, USA, Product Description SuperCos1 Cosmid Vektor Kit, code No. 251301), was dissociated with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code No. 27-0948-02) and also dephosphorylated with an alkaline shrimp phosphatase. Next, the cosmid DNA was dissociated with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code No. 27-0868-04). The cosmid DNA, treated in this manner, was mixed with the treated ATCC 13032 DNA, and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, No. code 27-0870-04). The binding mixture was then packaged in phages with the help of the Gigapack II XL Packaging Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code No. 200217). For infection of E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16: 1563-1575) the cells were collected in 10 mM MgSO4 and mixed with an aliquot of the phage suspension. Infection and titration of the cosmid bank were carried out as described in the quotation of Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the cells having been plated on an LB agar. (Lennox, 1955, Virology, 1: 190) with 100 mg / l ampicillin. After one overnight incubation at 37 ° C individual recombinant clones were selected.
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El ADN de cósmido de una colonia individual fue aislado con el estuche Qiaprep Spin Miniprep Kit (Nº de producto 27106, Qiagen, Hilden, Alemania) de acuerdo con los datos del fabricante, y disociado parcialmente con la enzima de restricción Sau3AI (Amersham Pharmacia, Freiburg, Alemania, Descripción del producto Sau3AI, Nº de producto 27-0913-02). Los fragmentos de ADN fueron desfosforilados con una fosfatasa alcalina de camarón (Roche Diagnostics GmbH, Mannheim, Alemania, Descripción del producto SAP, Nº de producto 1758250). Después de una separación por electroforesis en gel se efectuó el aislamiento de los fragmentos de cósmidos en el intervalo de tamaños de 1.500 a 2.000 pb (pares de bases = bps) con el estuche de extracción en gel QiaExII Gel Extraction Kit (Nº de producto 20021, Qiagen, Hilden, Alemania). El ADN del vector de secuenciación pZero-1, adquirido de la entidad Invitrogen (Groningen, Holanda, Descripción del producto Zero Background Cloning Kit, Nº de producto K2500-01) fue disociado con la enzima de restricción BamHI (Amersham Pharmacia, Freiburg, Alemania, Descripción del producto BamHI, Nº de producto 27-0868-04). La ligación de los fragmentos de cósmidos en el vector de secuenciación pZero-1 fue llevada a cabo tal como se describe por Sambrook y colaboradores (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) siendo incubada la mezcla de ADN durante una noche con la ligasa de T4 (Pharmacia Biotech, Freiburg, Alemania). Esta mezcla de ligación fue a continuación electroporada en la cepa de E. coli DH5\alphaMCR (Grant, 1990, Proceedings of the National Academy of Sciences EE.UU., 87:4645-4649) (Tauch y colaboradores 1994, FEMS Microbiol Letters, 123:343-7) y sembrada en placas sobre un agar LB (Lennox, 1955, Virology, 1:190) con 50 mg/l de zeocina. La preparación de los plásmidos de los clones recombinantes se efectuó con el Biorobot 9600 (Nº de producto 900200, Qiagen, Hilden, Alemania). La secuenciación se efectuó de acuerdo con el método de rotura de cadenas didesoxi de Sanger y colaboradores (1977, Proceedings of the National Academies of Sciences EE.UU., 74:5463-5467) con unas modificaciones de acuerdo con Zimmermann y colaboradores (1990, Nucleic Acids Research, 18:1067). Se utilizó el estuche de secuenciación "RR dRhodamin Terminator Cycle Sequencing Kit" von PE Applied Biosystems (Nº de producto 403044, Weiterstadt, Alemania). La separación por electroforesis en gel y el análisis de la reacción de secuenciación se efectuaron en un gel de "Rotiphorese NF Acrylamid/Bisacrylamid" (29:1) (Nº de producto A124.1, Roth, Karlsruhe, Alemania) con el aparato secuenciador "ABI Prism 377" de PE Applied Biosystems (Weiterstadt, Alemania).The cosmid DNA of an individual colony was insulated with the Qiaprep Spin Miniprep Kit case (Product No. 27106, Qiagen, Hilden, Germany) according to data from manufacturer, and partially dissociated with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Description of Sau3AI product, product no. 09-27-13-02). DNA fragments were dephosphorylated with an alkaline shrimp phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, SAP Product Description, Product No. 1758250). After a separation by gel electrophoresis isolation of the fragments of cosmids in the size range of 1,500 to 2,000 bp (pairs of bases = bps) with the QiaExII Gel gel extraction case Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). He DNA from the pZero-1 sequencing vector, acquired of the entity Invitrogen (Groningen, The Netherlands, Description of Product Zero Background Cloning Kit, Product No. K2500-01) was dissociated with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Description of BamHI product, product no. 08-28-04-04). The ligation of cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) the DNA mixture being incubated during one night with T4 ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was then electroporated in the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649) (Tauch et al 1994, FEMS Microbiol Letters, 123: 343-7) and plated on an LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l of zeocin The preparation of plasmids from clones recombinants was performed with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out of according to Sanger's dideoxy chain breakage method and collaborators (1977, Proceedings of the National Academies of Sciences USA, 74: 5463-5467) with some modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). The case of sequencing "RR dRhodamin Terminator Cycle Sequencing Kit" von PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany). Gel electrophoresis separation and analysis of the sequencing reaction was carried out on a gel of "Rotiphorese NF Acrylamid / Bisacrylamid" (29: 1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the sequencer "ABI Prism 377" by PE Applied Biosystems (Weiterstadt, Germany).
Los datos de secuencias en bruto que se obtuvieron fueron a continuación procesados mediando uso del paquete de programas de Staden ((1986, Nucleic Acids Research, 14:217-231) versión 97-0. Las secuencias individuales de los derivados de pZerol fueron ensambladas para formar un desfragmentador Contig coherente. El análisis de la zona de codificación, apoyado por ordenador, fue realizado con el programa XNIP (Staden, 1986, Nucleic Acids Research, 14:217-231). Otros análisis adicionales fueron llevados a cabo con los programas "BLAST search programs" (Altschul y colaboradores, 1997, Nucleic Acids Research, 25:3389-3402), frente al banco de datos no redundante del "National Center for Biotechnology Information" (NCBI, Bethesda, MD, EE.UU.).The raw sequence data to be they obtained were then processed mediating use of the package of Staden programs ((1986, Nucleic Acids Research, 14: 217-231) version 97-0. The individual sequences of pZerol derivatives were assembled to form a coherent contig defragmenter. He coding zone analysis, supported by computer, was carried out with the XNIP program (Staden, 1986, Nucleic Acids Research, 14: 217-231). Other additional analyzes were carried out with the programs "BLAST search programs "(Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402), in front of the data bank non-redundant of the "National Center for Biotechnology Information "(NCBI, Bethesda, MD, USA).
La secuencia de nucleótidos del gen dapC, que se obtuvo, se representa en SEQ ID No. 1. El análisis de la secuencia de nucleótidos proporcionó un cuadro de lectura abierto de 1.101 pares de bases, que fue designado como gen dapC. El gen dapC codifica un polipéptido de 367 aminoácidos, que está representado en SEQ ID No. 2.The nucleotide sequence of the dapC gene, which is obtained, is represented in SEQ ID No. 1. Sequence analysis of nucleotides provided an open reading frame of 1,101 base pairs, which was designated as dapC gene. The dapC gene encodes a 367 amino acid polypeptide, which is represented in SEQ ID No. 2.
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A partir de la cepa ATCC 13032 de acuerdo con el método de Eikmanns y colaboradores (Microbiology 140: 1817-1828 (1994)) se aisló un ADN cromosomal. En virtud de la secuencia del gen dapC, conocida a partir del Ejemplo 2 para C. glutamicum, se seleccionaron los siguientes oligonucleótidos para la reacción en cadena de la polimerasa (véanse también SEQ ID No. 5 y 6):From the strain ATCC 13032 according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) a chromosomal DNA was isolated. Under the sequence of the dapC gene, known from Example 2 for C. glutamicum , the following oligonucleotides were selected for the polymerase chain reaction (see also SEQ ID No. 5 and 6):
DapC (dCex1):DapC (dCex1):
- 5' GAT CTA (GAA TTC) GCC TCA GGC ATA ATC TAA CG 3'5 'GAT CTA (GAA TTC) GCC TCA GGC ATA ATC TAA CG 3 '
DapC (dCexna2):DapC (dCexna2):
- 5' GAT CTA (TCT AGA) CAG AGG ACA AGG CAA TCG GA 3'5 'CTA GAT (TCT AGA) CAG AGG ACA AGG CAA TCG GA 3 '
Los cebadores representados fueron sintetizados por la entidad ARK Scientific GmbH Biosystems (Darmstadt, Alemania) y la reacción de PCR se llevó a cabo de acuerdo con el método de PCR patrón de Innis y colaboradores (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) con la polimerasa Pwo de la entidad Roche Diagnostics GmbH (Mannheim, Alemania). Con ayuda de la reacción en cadena de la polimerasa, los cebadores hacen posible la amplificación de un fragmento de ADN con un tamaño de aproximadamente 1,6 kb (kilobases), que lleva el gen dapC. Además, el cebador DapC (dCex1) contiene la secuencia para el sitio de corte con la endonucleasa de restricción EcoRI, y el cebador DapC (dCexna2) contiene el sitio de corte con la endonucleasa de restricción XbaI, los cuales sitios están marcados mediante unos paréntesis en la secuencia de nucleótidos arriba representada.The primers represented were synthesized by the entity ARK Scientific GmbH Biosystems (Darmstadt, Germany) and the PCR reaction was carried out according to the PCR method Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with the Pwo polymerase of the Roche Diagnostics GmbH (Mannheim, Germany). With the help of polymerase chain reaction, primers make possible amplification of a DNA fragment with a size of approximately 1.6 kb (kilobases), which carries the dapC gene. In addition, the DapC primer (dCex1) contains the sequence for the site cutting with the EcoRI restriction endonuclease, and the primer DapC (dCexna2) contains the endonuclease cleavage site of XbaI restriction, which sites are marked by some parentheses in the nucleotide sequence shown above.
El fragmento de ADN amplificado, de aproximadamente 1,6 kb, que lleva el gen dapC, fue ligado con el estuche Zero Blunt® de la entidad Invitrogen Corporation (Carlsbad, CA, EE.UU.; número de catálogo K2700-20) en el vector pCR®Blunt II (Bernard y colaboradores, Journal of Molecular Biology, 234: 534-541 (1993)). A continuación la cepa Top10 de E. coli fue transformada con la tanda de ligación de acuerdo con los datos del fabricante del estuche (entidad Invitrogen Corporation, Carlsbad, CA, EE.UU.). La selección de células portadoras de plásmidos se efectuó por siembra en placas de la tanda de transformación sobre un agar LB (Sambrook y colaboradores, Molecular Cloning: A Laboratory Manual, 2ª edición, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), que había sido suplementada con 25 mg/l de kanamicina. El ADN de plásmido fue aislado a partir de un transformante con ayuda del estuche QIAprep Spin Miniprep Kit de la entidad Qiagen (Hilden, Alemania) y comprobado por tratamiento con las enzimas de restricción XbaI y EcoRI con una subsiguiente electroforesis en gel de agarosa (al 0,8%). La secuencia de ADN del fragmento de ADN amplificado fue comprobada por secuenciación. El plásmido fue denominado pCRdapC. La cepa fue designada como E. coli Top10/pCRdapC.The approximately 1.6 kb amplified DNA fragment carrying the dapC gene was ligated with the Zero Blunt® kit from the Invitrogen Corporation (Carlsbad, CA, USA; catalog number K2700-20) in the pCR® Blunt II vector (Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)). Then the Top10 strain of E. coli was transformed with the ligation batch according to the data of the case manufacturer (entity Invitrogen Corporation, Carlsbad, CA, USA). Plasmid carrier cell selection was performed by plating the transformation batch onto an LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 ), which had been supplemented with 25 mg / l kanamycin. Plasmid DNA was isolated from a transformant using the QIAprep Spin Miniprep Kit kit from Qiagen (Hilden, Germany) and checked by treatment with restriction enzymes XbaI and EcoRI with subsequent agarose gel electrophoresis (at 0.8%). The DNA sequence of the amplified DNA fragment was checked by sequencing. The plasmid was called pCRdapC. The strain was designated as E. coli Top10 / pCRdapC.
Como vector de partida para la construcción del vector de expresión de lanzadera de E. coli - C. glutamicum pEC-XT99A se utilizó el vector de expresión en E. coli pTRC99A (Amann y colaboradores 1988, Gene 69:301-315). Después de una disociación por restricción con BspHI (Roche Diagnostics GmbH, Mannheim, Alemania, Descripción del producto BspHI, Nº de producto 1467123) Produktbeschreibung BspHIy del subsiguiente tratamiento según Klenow (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto Klenow Fragment of DNA Polymerase I, Nº de producto 27-0928-01; método según Sambrook y colaboradores, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), se intercambió el gen de resistencia a ampicilina (bla) por el gen de resistencia a tetraciclina del plásmido de C. glutamicum pAG1 (Nº de acceso al banco de genes AF121000). Para esto, la región portadora del gen de resistencia se clonó como fragmento AluI (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto AluI, Nº de producto 27-0884-01) en el vector linealizado de expresión en E. Coli pTRC99A. La ligación se llevó a cabo tal como se ha descrito por Sambrook y colaboradores (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) siendo incubada la mezcla de ADN durante una noche con la ligasa de T4 (Pharmacia Biotech, Freiburg, Alemania). Esta mezcla de ligación fue electroporada (Tauch y colaboradores FEMS Microbiology Letters, 123:343-7) a continuación en la cepa de E. coli DH5\alphamcr (Grant, 1990, Proceedings of the National Academy of Sciences EE.UU., 87:4645-4649). El vector de expresión en E. coli construido fue designado por pXT99A.The E. coli pTRC99A expression vector was used as the starting vector for the construction of the E. coli shuttle vector - C. glutamicum pEC-XT99A (Amann et al 1988, Gene 69: 301-315). After a restriction dissociation with BspHI (Roche Diagnostics GmbH, Mannheim, Germany, Product Description BspHI, Product No. 1467123) Produktbeschreibung BspHI and the subsequent treatment according to Klenow (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description Klenow Fragment of DNA Polymerase I, Product No. 27-0928-01; method according to Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the ampicillin resistance (bla) gene was exchanged for the tetracycline resistance gene of the C. glutamicum pAG1 plasmid (Accession number to the gene bank AF121000). For this, the carrier region of the resistance gene was cloned as AluI fragment (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description AluI, Product No. 27-0884-01) in the linearized expression vector in E. Coli pTRC99A. Ligation was carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany ). This ligation mixture was electroporated (Tauch et al. FEMS Microbiology Letters, 123: 343-7) then in the E. coli strain DH5? (Grant, 1990, Proceedings of the National Academy of Sciences USA, 87 : 4645-4649). The expression vector in E. coli constructed was designated by pXT99A.
Como base para la clonación de un replicón mínimo procedente de Corynebacterium glutamicum, se utilizó el plásmido pGA1 (Sonnen y colaboradores 1991, Gene, 107:69-74). Mediante disociación por restricción con BalI/PstI (Promega GmbH, Mannheim, Alemania, Descripción del producto BalI, Nº de producto R6691; Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto PstI, Nº de producto 27-0976-01) del vector pGA1 se pudo clonar un fragmento con un tamaño de 3.484 pb en el vector pK18mob2 fragmentado con SmaI y PstI (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto SmaI, Nº de producto 27-0942-02, Descripción del producto PstI, Nº de producto 27-0976-01) (Tauch y colaboradores, 1998, Archives of Microbiology 169:303-312).As a basis for cloning a minimum replicon from Corynebacterium glutamicum , plasmid pGA1 was used (Sonnen et al. 1991, Gene, 107: 69-74). By restriction dissociation with BalI / PstI (Promega GmbH, Mannheim, Germany, Product Description BalI, Product No. R6691; Amersham Pharmacia Biotech, Freiburg, Germany, Product Description PstI, Product No. 27-0976-01) of the vector pGA1, a fragment with a size of 3,484 bp could be cloned into the vector pK18mob2 fragmented with SmaI and PstI (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description SmaI, Product No. 27-0942-02, Product Description PstI, No. of product 27-0976-01) (Tauch et al., 1998, Archives of Microbiology 169: 303-312).
Mediante disociación por restricción con BamHI/XhoI (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto BamHI, Nº de producto 27-086803, Descripción del producto XhoI, Nº de producto 27-0950-01) y por subsiguiente tratamiento según Klenow (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto Klenow Fragment of DNA Polymerase I, Nº de producto 27-0928-01; método según Sambrook y colaboradores, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) se delecionó (suprimió) un fragmento con un tamaño de 839 pb. A partir de la construcción artificial vuelta a ligar con la ligasa de T4 (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto T4-DNA-Ligase, Nº de producto 27-0870-04) se pudo clonar el replicón mínimo de C. glutamicum como un fragmento con un tamaño de 2.645 pb en el vector de expresión en E. coli pXT99A. Para esto el ADN de la construcción artificial portadora del replicón mínimo fue disociado con las enzimas de restricción KpnI (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto KpnI, Nº de producto 27-0908-01) y PstI (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto PstI, Nº de producto 27-0886-03) y a continuación mediante la polimerasa de Klenow (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto Klenow Fragment of DNA Polymerase I, Nº de producto 27-0928-01) se llevó a cabo un tratamiento con una 3'-5'-exonucleasa (Sambrook y colaboradores, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor).By restriction dissociation with BamHI / XhoI (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description BamHI, Product No. 27-086803, Product Description XhoI, Product No. 27-0950-01) and subsequent treatment according to Klenow ( Amersham Pharmacia Biotech, Freiburg, Germany, Product Description Klenow Fragment of DNA Polymerase I, Product No. 27-0928-01; method according to Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) was deleted (deleted ) a fragment with a size of 839 bp. From the artificial construction re-ligated with the T4 ligase (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description T4-DNA-Ligase, Product No. 27-0870-04) the minimum replicon of C. could be cloned . glutamicum as a fragment with a size of 2,645 bp in the expression vector in E. coli pXT99A. For this, the DNA of the artificial construction bearing the minimum replicon was dissociated with the restriction enzymes KpnI (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description KpnI, Product No. 27-0908-01) and PstI (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description PstI, Product No. 27-0886-03) and then by Klenow Polymerase (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description Klenow Fragment of DNA Polymerase I, Product No. 27-0928 -01) a 3'-5'-exonuclease treatment (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) was carried out.
En una tanda paralela, el vector de expresión en E. coli pXT99A se disoció con la enzima de restricción RsrII (Roche Diagnostics, Mannheim, Alemania, Descripción del producto RsrII, Nº de producto 1292587) y se preparó para la ligación con la polimerasa de Klenow (Amersham Pharmacia Biotech, Freiburg, Alemania, Klenow Fragment of DNA Polymerase I, Nº de producto 27-0928-01). La ligación del replicón mínimo con la construcción artificial de vector pXT99A se llevó a cabo tal como se ha descrito por Sambrook y colaboradores (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) siendo la mezcla de ADN incubada durante una noche con la ligasa de T4 (Amersham Pharmacia Biotech, Freiburg, Alemania, Descripción del producto T4-DNA-Ligase, Nº de producto 27-0870-04).In a parallel run, the E. coli pXT99A expression vector was dissociated with the restriction enzyme RsrII (Roche Diagnostics, Mannheim, Germany, Product Description RsrII, Product No. 1292587) and prepared for ligation with the polymerase of Klenow (Amersham Pharmacia Biotech, Freiburg, Germany, Klenow Fragment of DNA Polymerase I, Product No. 27-0928-01). The ligation of the minimum replicon with the artificial construction of vector pXT99A was carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) being the mixture of DNA incubated overnight with the T4 ligase (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description T4-DNA-Ligase, Product No. 27-0870-04).
El vector de expresión de lanzadera de E. coli - C. glutamicum pEC-XT99A, construido de esta manera, se transfirió mediante electroporación (Liebl y colaboradores, 1989, FEMS Microbiology Letters, 53:299-303) in C. glutamicum DSM5715. La selección de los transformantes se efectuó sobre un agar LBHIS, que se componía de 18,5 g/l de Brain-Heart Infusion Boullion (caldo de infusión de corazón-cerebro), 0,5 M de sorbitol, 5 g/l de Bacto-triptona, 2,5 g/l de extracto de Bacto-levadura, 5 g/l de NaCl y 18 g/l de Bacto-agar, que había sido suplementado con 5 mg/l de tetraciclina. La incubación se efectuó durante 2 días a 33ºC.The E. coli-C. glutamicum pEC-XT99A shuttle expression vector, constructed in this manner, was transferred by electroporation (Liebl et al., 1989, FEMS Microbiology Letters, 53: 299-303) in C. glutamicum DSM5715. Transformants were selected on an LBHIS agar, which consisted of 18.5 g / l of Brain-Heart Infusion Boullion (heart-brain infusion broth), 0.5 M sorbitol, 5 g / l of Bacto-tryptone, 2.5 g / l of Bacto-yeast extract, 5 g / l of NaCl and 18 g / l of Bacto-agar, which had been supplemented with 5 mg / l of tetracycline. The incubation was carried out for 2 days at 33 ° C.
El ADN de plásmido fue aislado a partir de un transformante de acuerdo con los métodos usuales (Peters-Wendisch y colaboradores, 1998, Microbiology, 144, 915-927), fue cortado con la endonucleasa de restricción HindIII y el plásmido fue comprobado por subsiguiente electroforesis en un gel de agarosa.Plasmid DNA was isolated from a transformant according to the usual methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), was cut with the HindIII restriction endonuclease and the plasmid was checked by subsequent electrophoresis in an agarose gel.
La construcción plasmídica así obtenida fue designada como pEC-XT99A y se representa en la Figura 1. La cepa obtenida por electroporación del plásmido pEC-XT99A en la cepa DSM5715 de Corynebacterium glutamicum fue denominada DSM5715/pEC-XT99A y depositada como DSM12967 en la Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Alemania) según el Convenio de Budapest.The plasmid construction thus obtained was designated as pEC-XT99A and is represented in Figure 1. The strain obtained by electroporation of plasmid pEC-XT99A in strain DSM5715 from Corynebacterium glutamicum was designated DSM5715 / pEC-XT99A and deposited as DSM12967 in Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) according to the Budapest Convention.
Como vector se utilizó el vector de lanzadera de E. coli - C. glutamicum pEC-XT99A, que se ha descrito en el Ejemplo 3.2. El ADN de este plásmido fue disociado completamente con las enzimas de restricción EcoRI y XbaI y a continuación desfosforilado con una fosfatasa alcalina de camarón (Roche Diagnostics GmbH, Mannheim, Alemania, Descripción del producto SAP, Nº de producto 1758250).As a vector, the E. coli-C. glutamicum pEC-XT99A shuttle vector was used, which has been described in Example 3.2. The DNA of this plasmid was completely dissociated with the restriction enzymes EcoRI and XbaI and then dephosphorylated with an alkaline shrimp phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, SAP Product Description, Product No. 1758250).
A partir del plásmido pCRdapC que se ha descrito en el Ejemplo 3.1., se aisló el gen dapC por disociación completa con las enzimas EcoRI y XbaI. El fragmento de dapC con un tamaño de aproximadamente 1.600 pb fue aislado a partir del gel de agarosa con el estuche QiaExII Gel Extraction Kit (Nº de producto 20021, Qiagen, Hilden, Alemania).From the plasmid pCRdapC described in Example 3.1., the dapC gene was isolated by complete dissociation with the enzymes EcoRI and XbaI. The dapC fragment with a size of approximately 1,600 bp was isolated from the agarose gel with the QiaExII Gel Extraction Kit case (Product No. 20021, Qiagen, Hilden, Germany).
El fragmento de dapC obtenido de este modo se mezcló con el vector pEC-XT99A previamente preparado y la tanda se trató con la ligasa de T4 (Amersham Pharmacia, Freiburg, Alemania, Descripción del producto T4-DNA-Ligase, Nº de código 27-0870-04). La tanda de ligación fue transformada en el seno de la cepa de E. coli DH5\alpha (Hanahan, In: DNA Cloning. A Practical Approach. Vol. I, IRL-Press, Oxford, Washington DC, EE.UU.). La selección de células portadoras del plásmido se efectuó mediante siembra en placas de la tanda de transformación sobre un agar de LB (Lennox, 1955, Virology, 1:190) con 5 mg/l de tetraciclina. Después de una incubación durante una noche a 37ºC se seleccionaron unos clones individuales recombinantes. El ADN de plásmido fue aislado a partir de un transformante con el estuche Qiaprep Spin Miniprep Kit (Nº de producto 27106, Qiagen, Hilden, Alemania) de acuerdo con los datos del fabricante, y fue disociado con las enzimas de restricción EcoRI y XbaI, con el fin de comprobar el plásmido mediante una subsiguiente electroforesis en gel de agarosa. El plásmido obtenido fue denominado pXT-dapCexp. Está representado en la Figura 2.The dapC fragment obtained in this way was mixed with the previously prepared pEC-XT99A vector and the batch was treated with T4 ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code No. 27- 0870-04). The ligation batch was transformed into the E. coli strain DH5α (Hanahan, In: DNA Cloning. A Practical Approach. Vol. I, IRL-Press, Oxford, Washington DC, USA). The selection of plasmid-bearing cells was carried out by plating the transformation batch on an LB agar (Lennox, 1955, Virology, 1: 190) with 5 mg / l of tetracycline. After an overnight incubation at 37 ° C, recombinant individual clones were selected. Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit kit (product no. 27106, Qiagen, Hilden, Germany) according to the manufacturer's data, and was dissociated with the restriction enzymes EcoRI and XbaI, in order to check the plasmid by a subsequent agarose gel electrophoresis. The plasmid obtained was called pXT-dapCexp. It is represented in Figure 2.
La cepa DSM5715 se transformó con el plásmido pXT-dapCexp mediante aplicación del método de electroporación que ha sido descrito por Liebl y colaboradores, (FEMS Microbiology Letters, 53:299-303 (1989)). La selección de los transformantes se efectuó sobre un agar LBHIS que se componía de 18,5 g/l del Brain-Heart Infusion Boullion, 0,5 M de sorbitol, 5 g/l de Bacto-triptona, 2,5 g/l de Bacto-extracto de levadura, 5 g/l de NaCl y 18 g/l de Bacto-agar, que había sido suplementado con 5 mg/l de tetraciclina. La incubación se efectuó durante 2 días a 33ºC.The DSM5715 strain was transformed with the plasmid pXT-dapCexp by applying the method of electroporation that has been described by Liebl et al., (FEMS Microbiology Letters, 53: 299-303 (1989)). The Transformants were selected on an LBHIS agar that it consisted of 18.5 g / l of the Brain-Heart Infusion Boullion, 0.5 M sorbitol, 5 g / l of Bacto-tryptone, 2.5 g / l of Bacto-yeast extract, 5 g / l NaCl and 18 g / l of Bacto-agar, which had been supplemented with 5 mg / l tetracycline. The incubation was carried out for 2 days at 33 ° C.
El ADN del plásmido se aisló a partir de un transformante de acuerdo con métodos usuales (Peters-Wendisch y colaboradores, 1998, Microbiology, 144, 915-927), se cortó con las endonucleasas de restricción EcoRI y XbaI, y el plásmido se comprobó por subsiguiente electroforesis en un gel de agarosa. La cepa obtenida fue denominada DSM5715/pXT-dapCexp y depositada como DSM 13254 en la Deutsche Sammlung für Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, Alemania) según el Convenio de Budapest.Plasmid DNA was isolated from a transformant according to usual methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), was cut with the restriction endonucleases EcoRI and XbaI, and the plasmid is checked by subsequent electrophoresis on an agarose gel. The strain obtained was named DSM5715 / pXT-dapCexp and deposited as DSM 13254 at the Deutsche Sammlung für Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) according to the Budapest Convention.
La cepa DSM5715/pXT-dapCexp de C. glutamicum, obtenida en el Ejemplo 4, fue cultivada en un medio nutritivo que es apropiado para la producción de lisina, y se determinó el contenido de lisina en el material sobrenadante del cultivo.The strain DSM5715 / pXT-dapCexp of C. glutamicum , obtained in Example 4, was grown in a nutrient medium that is appropriate for the production of lysine, and the lysine content in the culture supernatant material was determined.
Para esto la cepa se incubó primeramente durante 24 horas a 33ºC sobre una placa de agar con el correspondiente antibiótico (agar de Hirn-Herz (de cerebro-corazón) con tetraciclina (5 mg/l)). Partiendo de este cultivo en placas de agar, se inoculó un cultivo preliminar (10 ml del medio en un matraz Erlenmeyer de 100 ml de capacidad). Como medio para el cultivo preliminar se utilizó el medio completo CgIII.For this the strain was first incubated during 24 hours at 33 ° C on an agar plate with the corresponding antibiotic (Hirn-Herz agar (from brain-heart) with tetracycline (5 mg / l)). Starting from this culture in agar plates, a culture was inoculated preliminary (10 ml of the medium in a 100 ml Erlenmeyer flask of capacity). As a means for preliminary cultivation the complete medium CgIII.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
El valor del pH se ajustó a un pH de 7,4The pH value was adjusted to a pH of 7.4
A éste se le añadió tetraciclina (5 mg/l). El cultivo preliminar fue incubado sobre el aparato sacudidor a 240 rpm (revoluciones por minuto) durante 16 horas a 33ºC. De este cultivo preliminar se inoculó un cultivo principal, de manera tal que la densidad óptica (DO) inicial (a 660 nm) del cultivo principal fue de 0,1. Para el cultivo principal se utilizó el medio MM.To this was added tetracycline (5 mg / l). He preliminary culture was incubated on the shaker apparatus at 240 rpm (revolutions per minute) for 16 hours at 33 ° C. Of this preliminary culture a main crop was inoculated, so than the initial optical density (OD) (at 660 nm) of the main culture It was 0.1. The MM medium was used for the main culture.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Medio MMMeans, medium MM
El CSL (líquido de maceración de maíz), el MOPS y la solución de sal se ajustaron a un pH de 7 con agua amoniacal y se autoclavaron. A continuación se añadieron las soluciones estériles del substrato y de vitaminas, así como el CaCO_{3} autoclavado en seco.The CSL (corn maceration liquid), the MOPS and the salt solution was adjusted to a pH of 7 with ammonia water and They autoclaved. Then the solutions were added sterile substrate and vitamins, as well as CaCO_ {3} dry autoclaved
La cultivación se efectúa en un volumen de 10 ml en un matraz Erlenmeyer con una capacidad de 100 ml provisto de obstáculos. Se añadió tetraciclina (5 mg/l). La cultivación se efectuó a 33ºC y con una humedad del aire de 80%.Cultivation is carried out in a volume of 10 ml. in an Erlenmeyer flask with a capacity of 100 ml provided with obstacles Tetracycline (5 mg / l) was added. The cultivation is made at 33 ° C and with an air humidity of 80%.
Después de 72 horas se determinó la DO a una longitud de onda de medición de 660 nm con el aparato Biomek 1000 (de Beckmann Instruments GmbH, München). La cantidad formada de lisina se determinó con un aparato analizador de aminoácidos de la entidad Eppendorf-BioTronik (Hamburg, Alemania) mediante una cromatografía con intercambio de iones y mediante derivatización posterior en columna con detección por ninhidrina.After 72 hours the OD was determined at a 660 nm measuring wavelength with the Biomek 1000 (from Beckmann Instruments GmbH, München). The formed amount of lysine was determined with an amino acid analyzer apparatus of the Eppendorf-BioTronik entity (Hamburg, Germany) by ion exchange chromatography and by posterior column derivatization with detection by ninhydrin
En la Tabla 1 se representa el resultado del ensayoTable 1 shows the result of the test
Se adjuntan las siguientes Figuras:The following figures are attached:
Figura 1: mapa del plásmido pEC-XT99AFigure 1: plasmid map pEC-XT99A
Figura 2: mapa del plásmido pXT-dapCexpFigure 2: plasmid map pXT-dapCexp
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Las abreviaturas y denominaciones que se utilizan tienen los siguientes significados.The abbreviations and denominations that are They use have the following meanings.
- per: per:
- gen para el control de número de copias, procedente de pGA1gene for the control of number of copies, from from pGA1
- oriE: oriE:
- origen de replicación de E. coli codificado por un plásmido E. coli origin of replication encoded by a plasmid
- rep: rep:
- origen de replicación codificado por un plásmido, procedente del plásmido pGA1 de C. glutamicum origin of replication encoded by a plasmid, from plasmid pGA1 of C. glutamicum
- Ptrc: Ptrc:
- promotor trc procedente de pTRC99Atrc promoter from pTRC99A
- T1, T2: T1, T2:
- regiones de terminadores 1 y 2 procedentes de pTRC99Aterminator regions 1 and 2 from pTRC99A
- lacIq: lacIq:
- gen de represor del operón LacLac operon repressor gene
- Tet: Tet:
- gen de resistencia para tetraciclinaresistance gene for tetracycline
- dapC: dapC:
- gen dapC de C. glutamicum C. glutamicum dapC gene
- EcoRI: EcoRI:
- sitio de corte con la enzima de restricción EcoRIcutting site with restriction enzyme EcoRI
- EcoRV: EcoRV:
- sitio de corte con la enzima de restricción EcoRVcutting site with restriction enzyme EcoRV
- HindIII: HindIII:
- sitio de corte con la enzima de restricción HindIIIcutting site with restriction enzyme HindIII
- KpnI: KpnI:
- sitio de corte con la enzima de restricción KpnIcutting site with restriction enzyme KpnI
- SalI: I left:
- sitio de corte con la enzima de restricción SalIcutting site with restriction enzyme I left
- SmaI: SmaI:
- sitio de corte con la enzima de restricción SmaIcutting site with restriction enzyme SmaI
- NdeI: NdeI:
- sitio de corte con la enzima de restricción NdeIcutting site with restriction enzyme NdeI
- BamHI: BamHI:
- sitio de corte con la enzima de restricción BamHIcutting site with restriction enzyme BamHI
- NcoI: NcoI:
- sitio de corte con la enzima de restricción NcoIcutting site with restriction enzyme NcoI
- XbaI: XbaI:
- sitio de corte con la enzima de restricción XbaIcutting site with restriction enzyme XbaI
- SacI: SacI:
- sitio de corte con la enzima de restricción SacIcutting site with restriction enzyme SacI
\global\parskip0.000000\baselineskip\ global \ parskip0.000000 \ baselineskip
<110> Degussa-Huels AG<110> Degussa-Huels AG
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<120> Secuencias de nucleótidos que codifican el gen dapC y procedimiento para la preparación de L-lisina<120> Nucleotide sequences that encode the dapC gene and procedure for the preparation of L-lysine
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<130> 990217 BT<130> 990217 BT
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<140><140>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<141><141>
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<160> 6<160> 6
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<170> PatentIn Ver. 2.1<170> PatentIn Ver. 2.1
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 1<210> 1
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 1300<211> 1300
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<221> CDS<221> CDS
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<222> (91)..(1191)<222> (91) .. (1191)
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Gen dapC<223> dapC gene
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 1<400> 1
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 2<210> 2
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 367<211> 367
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> PRT<212> PRT
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 2<400> 2
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 3<210> 3
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 1300<211> 1300
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<221> CDS<221> CDS
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<222> (91)..(1191)<222> (91) .. (1191)
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> alelo dapC<223> dapC allele
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 3<400> 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 4<210> 4
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 367<211> 367
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> PRT<212> PRT
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 4<400> 4
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 5<210> 5
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 32<211> 32
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Descripción de la secuencia artificial: cebador<223> Sequence description artificial: primer
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Cebador DapC (dCex1)<223> DapC primer (dCex1)
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 5<400> 5
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgatctagaat tcgcctcagg cataatctaa cg
\hfill32
\ hskip-.1em \ dddseqskipgatctagaat tcgcctcagg cataatctaa cg
\ hfill32
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 6<210> 6
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 32<211> 32
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Descripción de la secuencia artificial: cebador<223> Sequence description artificial: primer
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Cebador DapC (dCexna2)<223> DapC primer (dCexna2)
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 6<400> 6
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgactatcta gacagaggac aaggcaatcg ga
\hfill32
\ hskip-.1em \ dddseqskipgactatcta gacagaggac aaggcaatcg ga
\ hfill32
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<110> Degussa-Huels AG<110> Degussa-Huels AG
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<120> Secuencias de nucleótidos que codifican el gen dapC y procedimiento para la preparación de L-lisina<120> Nucleotide sequences that encode the dapC gene and procedure for the preparation of L-lysine
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<130> 990217 BT<130> 990217 BT
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<140><140>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<141><141>
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<160> 6<160> 6
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<170> PatentIn Ver. 2.1<170> PatentIn Ver. 2.1
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 1<210> 1
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 1300<211> 1300
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<221> CDS<221> CDS
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<222> (91)..(1191)<222> (91) .. (1191)
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Gen dapC<223> dapC gene
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 1<400> 1
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 2<210> 2
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 367<211> 367
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> PRT<212> PRT
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 2<400> 2
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 3<210> 3
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 1300<211> 1300
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<221> CDS<221> CDS
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<222> (91)..(1191)<222> (91) .. (1191)
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Alelo de dapC<223> dapC allele
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 3<400> 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 4<210> 4
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 367<211> 367
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> PRT<212> PRT
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Corynebacterium glutamicum <213> Corynebacterium glutamicum
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 4<400> 4
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 5<210> 5
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 32<211> 32
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Descripción de la secuencia artificial: cebador<223> Sequence description artificial: primer
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Cebador DapC (dCex1)<223> DapC primer (dCex1)
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 5<400> 5
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgatctagaat tcgcctcagg cataatctaa cg
\hfill32
\ hskip-.1em \ dddseqskipgatctagaat tcgcctcagg cataatctaa cg
\ hfill32
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 6<210> 6
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 32<211> 32
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Descripción de la secuencia artificial: cebador<223> Sequence description artificial: primer
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<220><220>
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Cebador DapC (dCexna2)<223> DapC primer (dCexna2)
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 6<400> 6
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgatctatcta gacagaggac aaggcaatcg ga
\hfill32
\ hskip-.1em \ dddseqskipgatctatcta gacagaggac aaggcaatcg ga
\ hfill32
Claims (36)
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
- a) to)
- fermentación de una bacteria corineforme de acuerdo con las reivindicaciones 9 hasta 15;fermentation of a coryneform bacterium according with claims 9 to 15;
- b) b)
- enriquecimiento de la L-lisina en el medio o en las células de las bacterias, yenrichment of L-lysine in the medium or in the cells of bacteria, and
- c) C)
- aislamiento de la L-lisina.insulation of the L-lysine
- 17.1 el gen lysC que codifica una aspartato cinasa resistente a la retroalimentación,17.1 the lysC gene which encodes an aspartate kinase resistant to feedback,
- 17.2 el gen asd que codifica la aspartato-semialdehído deshidrogenasa,17.2 the asd gene that encodes aspartate-semialdehyde dehydrogenase,
- 17.3 el gen dapA que codifica la dihidrodipicolinato sintasa,17.3 the dapA gene which encodes dihydrodipicolinate synthase,
- 17.4 el gen dapB que codifica la dihidrodipicolinato reductasa,17.4 the dapB gene which encodes dihydrodipicolinate reductase,
- 17.5 el gen dapD que codifica la tetrahidrodipicolinato succinilasa,17.5 the dapD gene which encodes tetrahydrodipicolinate succinylase,
- 17.6 el gen dapE que codifica la N-succinil-diaminopimelato dessuccinilasa,17.6 the dapE gene that encodes the N-succinyl diaminopimelate dessuccinylase,
- 17.7 el gen dapF que codifica la diaminopimelato epimerasa17.7 the dapF gene encoding diaminopimelate epimerase
- 17.8 el gen lysA que codifica la diaminopimelato descarboxilasa,17.8 the lysA gene which encodes the diaminopimelate decarboxylase,
- 17.9 el gen ddh que codifica la diaminopimelato deshidrogenasa,17.9 the ddh gene which encodes diaminopimelate dehydrogenase,
- 17.10 el gen lysE que codifica la exportación de lisina,17.10 the gene lysE encoding the export of lysine,
- 17.11 el gen pyc que codifica la piruvato carboxilasa,17.11 the pyc gene which encodes pyruvate carboxylase,
- 17.12 el gen mqo que codifica la malato-quinona oxidorreductasa,17.12 the mqo gene which encodes the malate-quinone oxidoreductase,
- 17.13 el gen zwa1,17.13 the gene zwa1,
- 17.14 el gen gdh que codifica la glutamato deshidrogenasa.17.14 the gdh gene which encodes glutamate dehydrogenase.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10014546A DE10014546A1 (en) | 2000-03-23 | 2000-03-23 | Nucleotide sequences encoding the dapC gene and methods of producing L-lysine |
DE10014546 | 2000-03-23 |
Publications (1)
Publication Number | Publication Date |
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ES2344248T3 true ES2344248T3 (en) | 2010-08-23 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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ES01103850T Expired - Lifetime ES2344248T3 (en) | 2000-03-23 | 2001-02-16 | SEQUENCES OF NUCLEOTIDES CODING THE DAPC GENE, AND PROCEDURE FOR THE PREPARATION OF L-LISINE. |
Country Status (18)
Country | Link |
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US (2) | US6740742B2 (en) |
EP (1) | EP1136559B1 (en) |
JP (1) | JP2001299372A (en) |
KR (1) | KR20010090510A (en) |
CN (1) | CN100523193C (en) |
AT (1) | ATE465259T1 (en) |
AU (1) | AU2816301A (en) |
BR (1) | BR0101151B1 (en) |
CA (1) | CA2339307A1 (en) |
DE (2) | DE10014546A1 (en) |
DK (1) | DK1136559T3 (en) |
ES (1) | ES2344248T3 (en) |
HU (1) | HUP0101166A3 (en) |
ID (1) | ID29693A (en) |
MX (1) | MXPA01002860A (en) |
PT (1) | PT1136559E (en) |
SK (1) | SK3382001A3 (en) |
ZA (1) | ZA200102385B (en) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030157642A1 (en) * | 1997-07-15 | 2003-08-21 | Caldwell Robert M. | Increasing production of proteins in gram-positive microorganisms |
JP3965821B2 (en) * | 1999-03-09 | 2007-08-29 | 味の素株式会社 | Method for producing L-lysine |
US20020086371A1 (en) * | 1999-07-07 | 2002-07-04 | Degussa-Huls Aktiengesellschaft | L-lysine-producing corynebacteria and process for the preparation of L-lysine |
DE10101501A1 (en) * | 2001-01-12 | 2002-08-01 | Forschungszentrum Juelich Gmbh | Process for the fermentative production of pantothenic acid |
WO2006112000A1 (en) * | 2005-03-30 | 2006-10-26 | The Niigata Institute Of Science And Technology | Method of synthesizing 2-deoxy-scyllo-inosose by modified strain of e. coli, method of purifying the same and obtained 2-deoxy-scyllo-inosose |
EP3170889A1 (en) * | 2006-09-15 | 2017-05-24 | CJ Cheiljedang Corporation | A corynebacteria having enhanced l-lysine productivity and a method of producing l-lysine using the same |
KR100830826B1 (en) * | 2007-01-24 | 2008-05-19 | 씨제이제일제당 (주) | Process for producing fermentation product from carbon sources containing glycerol using corynebacteria |
US8932861B2 (en) | 2008-04-10 | 2015-01-13 | Cj Cheiljedang Corporation | Transformation vector comprising transposon, microorganisms transformed with the vector, and method for producing L-lysine using the microorganism |
KR101126041B1 (en) * | 2008-04-10 | 2012-03-19 | 씨제이제일제당 (주) | A transformation vector using transposon, a microorganism transformed with the vector and method of producing l-lysine using the microorganism |
GB2462645A (en) * | 2008-08-15 | 2010-02-17 | Advanced Technologies | Modification of plant threonine production by aspartate kinase |
DE102010019059A1 (en) * | 2010-05-03 | 2011-11-03 | Forschungszentrum Jülich GmbH | Sensors for intracellular metabolite detection |
WO2012056318A1 (en) * | 2010-10-28 | 2012-05-03 | Adisseo France S.A.S. | A method of production of 2,4-dihydroxybutyric acid |
CN102787106A (en) * | 2011-12-29 | 2012-11-21 | 西藏金稞集团有限责任公司 | Process for preparing glutamate dehydrogenase by fermentation method |
Family Cites Families (5)
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JPH0655149B2 (en) * | 1985-03-12 | 1994-07-27 | 協和醗酵工業株式会社 | Method for producing L-lysine |
JPH07155184A (en) * | 1993-12-08 | 1995-06-20 | Ajinomoto Co Inc | Production of l-lysine by fermentation method |
JP2004534501A (en) * | 1999-06-25 | 2004-11-18 | ビーエーエスエフ アクチェンゲゼルシャフト | Corynebacterium glutamicum gene encoding a metabolic pathway protein |
US20030049804A1 (en) * | 1999-06-25 | 2003-03-13 | Markus Pompejus | Corynebacterium glutamicum genes encoding metabolic pathway proteins |
JP4623825B2 (en) * | 1999-12-16 | 2011-02-02 | 協和発酵バイオ株式会社 | Novel polynucleotide |
-
2000
- 2000-03-23 DE DE10014546A patent/DE10014546A1/en not_active Withdrawn
-
2001
- 2001-02-16 PT PT01103850T patent/PT1136559E/en unknown
- 2001-02-16 AT AT01103850T patent/ATE465259T1/en active
- 2001-02-16 DK DK01103850.2T patent/DK1136559T3/en active
- 2001-02-16 ES ES01103850T patent/ES2344248T3/en not_active Expired - Lifetime
- 2001-02-16 EP EP01103850A patent/EP1136559B1/en not_active Expired - Lifetime
- 2001-02-16 DE DE50115438T patent/DE50115438D1/en not_active Expired - Lifetime
- 2001-03-12 SK SK338-2001A patent/SK3382001A3/en unknown
- 2001-03-19 MX MXPA01002860A patent/MXPA01002860A/en unknown
- 2001-03-21 AU AU28163/01A patent/AU2816301A/en not_active Abandoned
- 2001-03-21 CA CA002339307A patent/CA2339307A1/en not_active Abandoned
- 2001-03-22 KR KR1020010014868A patent/KR20010090510A/en not_active Application Discontinuation
- 2001-03-22 US US09/813,919 patent/US6740742B2/en not_active Expired - Lifetime
- 2001-03-22 HU HU0101166A patent/HUP0101166A3/en not_active Application Discontinuation
- 2001-03-22 ID IDP20010243D patent/ID29693A/en unknown
- 2001-03-22 JP JP2001083004A patent/JP2001299372A/en active Pending
- 2001-03-22 CN CNB011100117A patent/CN100523193C/en not_active Expired - Fee Related
- 2001-03-22 ZA ZA200102385A patent/ZA200102385B/en unknown
- 2001-03-23 BR BRPI0101151-0B1A patent/BR0101151B1/en not_active IP Right Cessation
-
2003
- 2003-12-29 US US10/745,684 patent/US7759056B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
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SK3382001A3 (en) | 2001-12-03 |
ID29693A (en) | 2001-09-27 |
DK1136559T3 (en) | 2010-08-02 |
US20050100927A1 (en) | 2005-05-12 |
EP1136559A3 (en) | 2003-12-10 |
DE50115438D1 (en) | 2010-06-02 |
MXPA01002860A (en) | 2002-08-06 |
DE10014546A1 (en) | 2001-09-27 |
HU0101166D0 (en) | 2001-05-28 |
CA2339307A1 (en) | 2001-09-23 |
HUP0101166A2 (en) | 2003-08-28 |
EP1136559A2 (en) | 2001-09-26 |
ATE465259T1 (en) | 2010-05-15 |
US7759056B2 (en) | 2010-07-20 |
ZA200102385B (en) | 2001-09-26 |
BR0101151A (en) | 2001-10-30 |
US6740742B2 (en) | 2004-05-25 |
EP1136559B1 (en) | 2010-04-21 |
KR20010090510A (en) | 2001-10-18 |
BR0101151B1 (en) | 2014-12-30 |
AU2816301A (en) | 2002-07-25 |
PT1136559E (en) | 2010-07-14 |
CN100523193C (en) | 2009-08-05 |
HUP0101166A3 (en) | 2005-12-28 |
US20010049123A1 (en) | 2001-12-06 |
JP2001299372A (en) | 2001-10-30 |
CN1319668A (en) | 2001-10-31 |
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